Modified carbocyanine dyes and their conjugates

ABSTRACT

Chemically reactive carbocyanine dyes incorporating an indolium ring moiety that is substituted at the 3-position by a reactive group or by a conjugated substance, and their uses, are described. Conjugation through this position results in spectral properties that are uniformly superior to those of conjugates of spectrally similar dyes wherein attachment is at a different position. The invention includes derivative compounds having one or more benzo nitrogens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/150,596, filedJun. 10, 2005, which is a divisional of U.S. Ser. No. 09/968,401 filedSep. 29, 2001 (U.S. Pat. No. 6,977,305) and U.S. Ser. No. 09/969,853,filed Oct. 1, 2001 (U.S. Pat. No. 6,974,873), which claims priority toU.S. Ser. No. 60/236,637, filed Sep. 29, 2000; and U.S. Ser. No.60/276,870, filed Mar. 16, 2001, which disclosures are hereinincorporated by reference.

FIELD OF THE INVENTION

The invention relates to colored and fluorescent chemicals, includingreactive dyes and dye-conjugates; and to their uses.

BACKGROUND OF THE INVENTION

Fluorescent compounds are covalently or noncovalently attached to othermaterials to impart color and fluorescence. Brightly fluorescent dyespermit detection or location of the attached materials with greatsensitivity. Certain carbocyanine dyes have demonstrated utility aslabeling reagents for a variety of biological applications, e.g. U.S.Pat. Nos. 4,981,977 to Southwick, et al. (1991); 5,268,486 to Waggoneret al. (1993); 5,569,587 to Waggoner (1996); 5,569,766 to Waggoner etal. (1996); 5,486,616 to Waggoner et al. (1996); 5,627,027 to Waggoner(1997); 5,808,044 to Brush, et al. (1998); 5,877,310 to Reddington, etal. (1999); 6,002,003 to Shen, et al. (1999); 6,004,536 to Leung et al.(1999); 6,008,373 to Waggoner, et al. (1999); 6,043,025 to Minden, etal. (2000); 6,127,134 to Minden, et al. (2000); 6,130,094 to Waggoner,et al. (2000); 6,133,445 to Waggoner, et al. (2000); also WO 97/40104,WO 99/51702, WO 01/21624, and EP 1 065 250 A1; and TETRAHEDRON LETTERS41, 9185-88 (2000); all of the above incorporated by reference.Nevertheless, many carbocyanine dyes are known to share certaindisadvantages, e.g. severe quenching of the fluorescence of carbocyaninedyes in biopolymer conjugates, e.g. quenching of Cy5 and Cy7 dyevariants on conjugates, as discussed by Gruber et al., BIOCONJUGATECHEM. 11, 696 (2000), and in EP 1 065 250 A1, 0004. In addition, certaindesired sulfoalkyl derivatives of the reactive carbocyanine dyes aredifficult to prepare, as indicated for Cy3 and Cy5 variants by Waggonerand colleagues in BIOCONJUGATE CHEM. 4, 105, 109 col. 2 (1993). Cyaninedyes also have a very strong tendency to self-associate (i.e. stack),which can significantly reduce the fluorescence quantum yields, asdescribed in the extensive review by Mishra, et al., CHEM. REV. 100,1973 (2000).

Modification of an indolium ring of the carbocyanine dye to permit areactive group or conjugated substance at the number 3 positionunexpectedly mitigates these problems and results in dye-conjugates thatare uniformly and substantially more fluorescent on proteins, nucleicacids and other biopolymers, than conjugates labeled with structurallysimilar carbocyanine dyes bound through the nitrogen atom at the number1 position. In addition to having more intense fluorescence emissionthan structurally similar dyes at virtually identical wavelengths, anddecreased artifacts in their absorption spectra upon conjugation tobiopolymers, certain embodiments of the invention also have greaterphotostability and higher absorbance (extinction coefficients) at thewavelength(s) of peak absorbance than such structurally similar dyes.These improvements result in significantly greater sensitivity in assaysthat use these dyes and their conjugates, while utilizing availablefilters and instrumentation already commercially available for use withsimilar dyes.

Furthermore, the dyes of the invention typically exhibit absorbancemaxima between about 530 nm and about 800 nm, so dyes can be selected tomatch the principal emission lines of the mercury arc lamp (546 nm),frequency-doubled Nd-Yag laser (532 nm), Kr-ion laser (568 nm and 647nm), HeNe laser (543 nm, 594 nm, and 633 nm) or long-wavelength laserdiodes (especially 635 nm and longer). The azacarbocyanine dyes of theinvention exhibit a bathochromic spectral shift (a shift to longerwavelength) of approximately 20 to 50 nm relative to otherwisestructurally similar carbocyanine dyes known in the art. Some dyes ofthe invention exhibit very long wavelength excitation (at least 640 nm,but some greater than about 730 nm) and emission bands (at least 665 nm,and some greater than about 750 nm), so they are particularly useful forsamples that are transparent to infrared wavelengths.

DESCRIPTION OF DRAWINGS

FIG. 1. Fluorescence excitation and emission spectra of 1.0 μM solutions(pH=7.5) of Cy5 and Compound 8 are shown. Both the excitation andemission characteristics of the Compound 8 are very similar to those ofCy5, when present as the free-acid.

FIG. 2. Comparison of the absorption and emission spectra of Cy5 andCompound 9 when conjugated to Goat Anti-Mouse IgG (GAM) at DOS of ˜4.8and 4.2 respectively (see Example 68).

FIG. 3. Comparison of the absorption/excitation spectra of Compound 9GAM and Cy5 derivatized GAM. Excitation spectra (emission wavelength=725nm) normalized to absorbance spectra at 660 nm (see Example 69).

FIG. 4. Direct comparison of absorbance properties of Compound 30(Cy5-type linkage, open circles) with Compound 24 (of the invention,closed circles) when conjugated to GAR at DOS's of approximately 2.8,4.3, and 5.5 (600 nm absorbance bands increasing as a function ofincreasing DOS, 650 nm absorbance bands normalized to 1.0) (see Example70).

FIG. 5. Flow cytometric comparison of Compound 9 labeled GAM with thecommercially available Cy5 GAM from Jackson Labs and Amersham-PharmaciaBiotech (see Example 72).

FIG. 6. Comparison of the fluorescence of goat anti-rabbit IgG (GAR)conjugates of Compound 13 (solid line) and those of the spectrallysimilar CY3 dye (dashed line) (see Example 73).

FIG. 7. Photostability comparison of Compound 9 (solid circles) with Cy5(open circles), (40× objective, pH=7.5 solution) (see Example 74).

FIG. 8. Fluorescence energy-transfer from R-phycoerythrin to Compound 9in a tandem conjugate (excitation=488 nm). Donor alone (open circles,left y-axis), donor-acceptor pair (closed circles, right y-axis) (seeExample 76).

FIG. 9. Fluorescence energy-transfer from Allophycocyanin to Compound 22in a tandem conjugate (excitation=633 nm). Donor alone (open circles,left y-axis), donor-acceptor pair (closed circles, right y-axis) (seeExample 76).

FIG. 10. Flow cytometric comparison of Streptavidin-(R-phycoerythrin)(SA-(R-PE)) conjugates of Compound 9 and Cy5 when targeted to a cellsurface CD3 marker (see Example 77).

FIG. 11. Comparison of the change in absorbance of Compound 9 (closedcircles) and Cy5 (open circles) upon DNA incorporation using ULSmethodology (see Example 92).

FIG. 12. Comparison of the absorption and fluorescence properties ofidentical concentrations of Compound 9 (solid line) and Cy5 (dashedline) labeled cDNA (see Example 94).

FIG. 13. Change in absorbance of Cy5 derivatives of cDNA upon digestionwith micrococcal nuclease. Approximately one Cy5 dye for every 23 basesof DNA (see Example 94).

SUMMARY OF THE INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention describes modified carbocyanine dyes and theirconjugates. Preferred compounds have at least one substituted indoliumring system wherein the substituent on the 3-carbon of the indolium ringcontains a chemically reactive group or a conjugated substance. Otherpreferred compounds incorporate an azabenzazolium ring moiety and atleast one sulfonate moiety. The dyes and dye conjugates are used tolocate or detect the interaction or presence of analytes or ligands in asample. Kits incorporating such dyes or dye conjugates facilitate theiruse in such methods.

Dyes

The carbocyanine dyes of the invention typically comprise twoheterocyclic ring systems bound together by a polymethine linker,according to the formula:A-BRIDGE-Bwhere A is a first heterocyclic ring system that is a substitutedbenzazolium ring that optionally incorporates one or more nitrogen atoms(azabenzazolium rings), B is a second heterocyclic ring system that is asubstituted benzazolium or azabenzazolium ring, and BRIDGE is apolymethine linker that is optionally substituted. The first and secondring systems and polymethine linker are optionally further substitutedby a variety of substituents or are fused to additional rings that areoptionally further substituted. In one aspect of the invention, thecarbocyanine dye contains a chemically reactive group or a conjugatedsubstance that is attached at carbon 3 of an indolium ring system. In apreferred embodiment, the carbocyanine dye is further substituted one ormore times by sulfo or sulfoalkyl.

By “sulfo” is meant sulfonic acid, or salt of sulfonic acid (sulfonate).Similarly, by “carboxy” is meant carboxylic acid or salt of carboxylicacid. “Phosphate”, as used herein, is an ester of phosphoric acid, andincludes salts of phosphate. “Phosphonate”, as used herein, meansphosphonic acid and includes salts of phosphonate. As used herein,unless otherwise specified, the alkyl portions of substituents such asalkyl, alkoxy, arylalkyl, alkylamino, dialkylamino, trialkylammonium, orperfluoroalkyl are optionally saturated, unsaturated, linear orbranched, and all alkyl, alkoxy, alkylamino, and dialkylaminosubstituents are themselves optionally further substituted by carboxy,sulfo, amino, or hydroxy.

The A moiety has the formula:

wherein Y represents the atoms necessary to form one to two fusedaromatic rings having 6 atoms in each ring, which atoms are selectedfrom —CH, —C, —CR¹, and —N(R²), where each β is 0 or 1 (such that eachring nitrogen is either quaternized or not), and each R¹ isindependently -L-R_(x); or -L-S_(x); or amino, sulfo, trifluoromethyl,or halogen; or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂dialkylamino, optionally further substituted. Incorporation of one ormore non-hydrogen substituents on the fused rings can be used to finetune the absorption and emission spectrum of the resulting dye. In oneembodiment, there is at least one non-hydrogen substituent, preferablysulfo, an alkoxy or halogen; preferably the halogen is bromine. In oneembodiment, R¹ is independently -L-R_(x); or -L-S_(c); or amino, sulfo,trifluoromethyl, or halogen; or C₁-C₆ alkyl, which is optionally furthersubstituted by carboxy, sulfo, amino, or hydroxy.

In one embodiment, X is one of O, S, Se or NR⁵, where R⁵ is H or analkyl group having 1-22 carbons, that is optionally substituted one ormore times by hydroxy, carboxy, sulfo, amino, alkylamino having 1-6carbons or dialkylamino having 2-12 carbons. Alternatively, X is O, S,or —CR³R⁴, where R³ and R⁴, which may be the same or different, arealkyl or arylalkyl, and optionally further substituted. Preferably, R³is -L-R_(x) or -L-S_(c) (as defined below).

The substituents R², R⁴, and R¹² are independently -L-R_(x); or-L-S_(c); or a C₁-C₂₂ alkyl or C₇-C₂₂ arylalkyl, each alkyl portion ofwhich optionally incorporates up to six hetero atoms, selected from N, Oand S, and each alkyl portion of which is optionally substituted one ormore times by fluorine, chlorine, bromine, iodine, hydroxy, carboxy,sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido,C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino, or C₃-C₁₈trialkylammonium; or R³ and R⁴ taken in combination complete a five- orsix-membered saturated or unsaturated ring that is substituted by-L-R_(x); or -L-S_(c). Preferably, R⁴ is alkyl having 1-6 carbons,optionally substituted one or more times by fluorine, chlorine, bromine,iodine, hydroxy, carboxy, sulfo, or amino; more preferably R⁴ is methylor ethyl. In one aspect of the invention, R⁴ is methyl. Alternatively,R⁴ in combination with R²¹ forms a 6-membered ring, as described below;or R⁴ taken in combination with R³ forms a saturated or unsaturated ringsubstituent, that is substituted by -L-R_(x) or -L-S_(c).

The substitutents R² and R¹² are typically independently -L-R_(x); or-L-S_(c); or a C₁-C₆ alkyl, which is optionally substituted one or moretimes by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino. Preferably R²and R¹² are independently alkyl with 1-6 carbon atoms that areunsubstituted or are substituted once by hydroxy, sulfo, carboxy oramino. Where either R² or R¹² is substituted by hydroxy, sulfo, carboxyor amino, the substituent is preferably separated from the indolium orother benzazolium nitrogen atom by 2-6 carbon atoms. Where R² and R¹²are unsubstituted alkyl groups, they are preferably methyl or ethyl,most preferably methyl. Typically R² and R¹² are the same and aremethyl, ethyl, sulfopropyl or sulfobutyl.

The B moiety has the formula:

where W represents the atoms necessary to form one to two fused aromaticrings having 6 atoms in each ring, which atoms are selected from —CH,—C, —CR^(1′), and —N(R¹²)_(β′), where each β′ is 0 or 1 (such that eachring nitrogen is either quaternized or not), and each R^(1′) isindependently -L-R_(x); or -L-S_(c); or amino, sulfo, trifluoromethyl,or halogen; or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂dialkylamino, each of which is optionally further substituted bycarboxy, sulfo, amino, or hydroxy. Where the six membered rings form anazabenzazole ring system, they typically incorporate 1-3 nitrogen atoms,more preferably 1-2 nitrogen atoms, typically incorporated in the first6-membered aromatic ring fused to the azole ring. In one aspect of theinvention, the ring system W contains only carbon atoms and is abenzazole ring system. In one embodiment, each R^(1′) is independently-L-R_(x); or -L-S_(c); or amino, sulfo, trifluoromethyl, or halogen; orC₁-C₆ alkyl, which is optionally further substituted by carboxy, sulfo,amino, or hydroxy.

Where A or B is an azabenzazolium, the fused aromatic rings typicallyincorporate 1-3 nitrogen atoms, more preferably 1-2 nitrogen atoms,typically incorporated in the first 6-membered aromatic ring fused tothe azole ring. Preferred embodiments of the azabenzazole moiety includewithout limitation the following structures, (and the equivalentstructures where the nitrogen is quaternized by R¹²):

When Y or W includes a nitrogen atom, at least one of the azabenzazolenitrogen atoms is quaternized, resulting in a formal positive charge. Inone embodiment, the azole nitrogen atom is quaternized, and the benzonitrogen atom is unsubstituted. Preferably, the azole nitrogen atom isunsubstituted and at least one benzo nitrogen atom is quaternized.Typically, no more than one azole nitrogen on a given azabenzazole isquaternized, i.e. α is 0 or 1, β is 0 or 1, and α+all β=1; and δ is 0 or1, β′ is 0 or 1, and δ+all β′=1. The presence of additional fused6-membered rings (as in Formula VI, above) shift the wavelength evenfurther.

Choice of the X and Z moieties may also affect the dye's absorption andfluorescence emission properties. X and Z are optionally the same ordifferent, and spectral properties of the resulting dye may be tuned bycareful selection of X and Z. In one embodiment, Z is one of O, S, Se orNR¹⁵, where R¹⁵ is H or an alkyl group having 1-22 carbons, that isoptionally substituted one or more times by hydroxy, carboxy, sulfo,amino, alkylamino having 1-6 carbons or dialkylamino having 2-12carbons. Alternatively, Z is O, S, or —CR¹³R¹⁴, where R¹³ and R¹⁴, whichmay be the same or different, are alkyl or arylalkyl, and optionallyfurther substituted. Typically X and Z are —CR³R⁴ and —CR¹³R¹⁴,respectively.

Where Z is —CR¹³R¹⁴, the substituents R¹³ and R¹⁴, which may be same ordifferent, are independently -L-R_(x); or -L-S_(c); or a C₁-C₂₂ alkyl orC₇-C₂₂ arylalkyl, each alkyl portion of which optionally incorporates upto six hetero atoms, selected from N, O and S, and each alkyl portion ofwhich is optionally substituted one or more times by fluorine, chlorine,bromine, iodine, hydroxy, carboxy, sulfo, phosphate, amino, sulfate,phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, orC₂-C₁₂ dialkylamino, or C₃-C₁₈ trialkylammonium. Alternatively, R¹³ andR¹⁴ in combination complete a five or six membered saturated orunsaturated ring that is optionally substituted by -L-R_(x); or-L-S_(c); or R¹³ or R¹⁴ combines with a methine substituent to form aring, as described below. In one embodiment, one of R¹³ and R¹⁴ is aC₁-C₆ alkyl and the other of R¹³ and R¹⁴ is -L-R_(x); or -L-S_(c); or aC₁-C₂₂ alkyl, which is optionally substituted one or more times by F,Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate,phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, orC₂-C₁₂ dialkylamino. Preferably R¹³ and R¹⁴ are independently alkyl with1-6 carbon atoms that are unsubstituted or are substituted once byhydroxy, sulfo, carboxy or amino. Where either R¹³ or R¹⁴ is substitutedby hydroxy, sulfo, carboxy or amino, the substituent is preferablyseparated from the indolium or other benzazolium nitrogen atom by 2-6carbon atoms. In one aspect of the invention, R¹³ and R¹⁴ are alkylshaving 1-6 carbons, preferably methyls. In another aspect of theinvention, one of R¹³ and R¹⁴ is methyl, and the other is alkyl having1-6 carbons that is substituted by carboxy or by sulfo or by hydroxy, orby -L-R_(X) or -L-S_(C).

The BRIDGE moiety has the formula:

wherein a and b are independently 0 or 1. In a preferred aspect of theazacarbocyanine dyes, either a or b is 1, not both. The length of thepolymethine bridge between the heterocyclic ring systems, also affectsthe dye's absorption and emission properties. Where Z is CR¹³R¹⁴, and aand b=0, and the indolium heterocycle is not fused to additional rings,the resulting “indocarbocyanine” dye typically exhibits an absorptionmaximum near 550 nm. Where a=1 and b=0, the “indodicarbocyanines” dyestypically absorb maximally near 650 nm. The “indotricarbocyanine” dyes,where a and b are both 1, typically absorbs maximally near 750 nm.

Each of R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, and R²⁷, when present, isindependently H, F, Cl, alkyl having 1-6 carbons, alkoxy having 1-6carbons, aryloxy, a N-heteroaromatic moiety, or an iminium ion.Alternatively, two substituents R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, and R²⁷,when taken in combination, form a 4-, 5-, or 6-membered saturated orunsaturated hydrocarbon ring that is unsubstituted or is optionallysubstituted one or more times by a saturated or unsaturated alkyl having1-6 carbons, halogen, or a carbonyl oxygen, or thiocarbonyl. In yetanother embodiment, R²¹ combines with R⁴ to form a 6-membered ring thatis optionally substituted by alkyl having 1-6 carbons. Alternatively,R²³ (where a and b are both 0), R²⁵ (where a=1 and b=0), or R²⁶ (where aand b are both 1) taken in combination with one of R¹³ and R¹⁴ forms a6-membered ring that is optionally substituted by alkyl having 1-6carbons.

Examples of appropriate BRIDGE moieties have been previously describedin the literature, including BRIDGE moieties that incorporatenonhydrogen substituents, ring structures, and rigidizing elements (U.S.Pat. Nos. 5,831,098 to Ollmann, Jr (1998); 6,086,737 to Patonay et al.(2000); 6,048,982 to Waggoner (2000); and 5,453,505 to Lee et al.(1995); 5,639,874 to Middendorf et al. (1997); 3,864,644 to Lincoln etal (1975); 4,011,086 to Simson (1977); all incorporated by reference).

Typically, each of R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, and R²⁷, when present,is H. Where one of R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, and R²⁷ is nonhydrogen,it is typically the substituent on the center carbon of BRIDGE, i.e.,R²² where a=0 and b=0, R²³ were a=1 and b=0, and R²⁴ where a=1 and b=1.Similarly, where BRIDGE incorporates a 4-, 5-, or 6-membered ring, ittypically occurs at the center of the BRIDGE moiety, for instance asshown below for a pentamethine dye:

A preferred version of the invention is a compound of the formula:

and its salts, where R², R³, R⁴, R¹², α, δ, W, Y, and Z are as definedpreviously. For simplicity, R²¹⁻²³ are independently as definedpreviously for R²¹⁻²⁷, and n=1, 2, or 3. Where n is >3, the dyes havespectra even further shifted into the infrared region. More preferably,the dye has the formula:

The substituents R⁶-R⁹ are independently selected from H, alkyl havingfrom 1-6 carbons, alkoxy having 1-6 carbons, amino, alkylamino having1-6 carbons, or dialkylamino having 2-12 carbons, sulfo, carboxy,perfluoroalkyl having 1-6 carbons, or halogen.

In one aspect of the invention, both A and B are benzazolium rings,according to the formula:

where the substituents R¹⁶-R¹⁹ are independently selected from H, alkylhaving from 1-6 carbons, alkoxy having 1-6 carbons, amino, alkylaminohaving 1-6 carbons, or dialkylamino having 2-12 carbons, sulfo, carboxy,perfluoroalkyl having 1-6 carbons, or halogen.

Incorporation of one or more non-hydrogen substituents on either or bothbenzazolium rings are useful to fine-tune the absorption and emissionspectrum. There is typically at least one non-hydrogen substituent oneach of the benzazolium rings, preferably sulfo, an alkoxy or a halogensubstituent. Typically, the substituents on the benzo rings are H orsulfo. In one embodiment, one of R⁶, R⁷, R⁸, and R⁹ or of R¹⁶, R¹⁷, R¹⁸,and R¹⁹ is a dialkylamino that is a saturated 5- or 6-membered nitrogenheterocycle, such as piperidine. Additionally, any two adjacentsubstituents of R⁶-R⁹ and R¹⁶-R¹⁹ are optionally taken in combination toform one or more fused aromatic rings. These additional rings areoptionally further substituted as described above for R⁶-R⁹ and R¹⁶-R¹⁹,and in particular by sulfonic acids.

Selected examples of carbocyanine dyes of the invention possessingadditional fused aromatic rings are given below (for simplicity, all buta few of the possible substituents are shown as hydrogen, with theshortest polymethine bridge):

These basic structures, and their longer-wavelength analogs, areoptionally further substituted as described in this section. Additionalvariants not specifically depicted above are also within the scope ofthe invention, e.g. the “left-hand” indolium of Formula XVIII linked toa benzazolium of Formula XVII, XIX, or XX; azabenzazolium versions, suchas

and variations thereof, according to the formulas as above.

In another aspect of the invention, A is a benzazolium and B is anazabenzazolium, according to the formula:

where the substituents R⁶-R⁹ and R¹⁶-R¹⁹ are as described previously.Preferred substitutents for R¹⁶ through R¹³ are independently H,-L-R_(x); or -L-S_(c); or amino, trifluoromethyl, or halogen; or C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each ofwhich is optionally further substituted by carboxy, sulfo, amino, orhydroxy. Preferably, the only non-hydrogen substituent on the B side,when present, is a bromine at preferably bromine at R¹⁷. Preferably n=2.

In one aspect of the invention, the carbocyanine dyes of the inventionare sulfonated one or more times. If the dye of the invention issubstituted by sulfo, it is typically sulfonated at R⁷ or R¹⁷ or both,or sulfoalkylated at R² or R¹² or both, or is both sulfonated andsulfoalkylated. Typically, where the aromatic ring of Y or W containsone or more nitrogen atoms, the ring with the nitrogen is notsulfonated. Generally, commercially available reactive carbocyanine dyesare sulfonated up to two times (at positions corresponding to R⁷ andR¹⁷, and as sulfoalkyl at one of R² and R¹²), leaving one of R² and R¹²for the location of the reactive group. In contrast, by attaching thereactive group (or conjugated substance) at R³, certain carbocyaninedyes of the invention may be sulfonated at least four times (at R⁷, atR¹⁷, and as sulfoalkyl at R² and R¹²). This extra sulfonation, as wellas the change in attachment site, results in reactive dyes and dyeconjugates that are brighter, more soluble in aqueous solutions, andmore resistant to the fluorescence quenching that results from dye-dyestacking interactions. However, sulfonation by four or more sulfonicacids is not required for the dyes of the invention to have spectralproperties that are superior to those of structurally similar dyes thatare not linked through the 3 position of the indolium ring (FIG. 4).

In addition, the dyes of the invention are substituted by one or morechemically reactive groups (-L-R_(x)) or conjugated substances(-L-S_(C)), as described below. Typically, the -L-R_(x) or -L-S_(C)moieties are bound to the dye at an R², R³, R⁴, R¹³ or R¹⁴.Alternatively, -L-R_(x) or -L-S_(C) is bound to the dye at an aromaticcarbon atom of the azabenzazolium ring, or the benzazolium ring. In apreferred embodiment, one or more of R² and R¹² is -L-R_(x) or -L-S_(C).In yet another preferred embodiment of the invention, one or more of R³,R⁴, R¹³, and R¹⁴ is -L-R_(x) or -L-S_(C). Alternatively, one or more ofR²¹, R²², R²³, R²⁴, R²⁵, R²⁶, and R²⁷ is L-R_(x) or -L-S_(C). In apreferred embodiment, the dye of the invention is substituted by onlyone -L-R_(x) or -L-S_(C).

Many embodiments of the compounds of the invention possess an overallelectronic charge. It is to be understood that when such electroniccharges are shown to be present, they are balanced by the presence of anappropriate counterion, which may or may not be explicitly identified. Abiologically compatible counterion, which is preferred for someapplications, is not toxic in biological applications, and does not havea substantially deleterious effect on biomolecules. Where the compoundof the invention is positively charged, the counterion is typicallyselected from, but not limited to, chloride, bromide, iodide, sulfate,alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylboride, nitrate and anions of aromatic oraliphatic carboxylic acids. Where the compound of the invention isnegatively charged, the counterion is typically selected from, but notlimited to, alkali metal ions, alkaline earth metal ions, transitionmetal ions, ammonium or substituted ammonium or pyridinium ions.Preferably, any necessary counterion is biologically compatible, is nottoxic as used, and does not have a substantially deleterious effect onbiomolecules. Counterions are readily changed by methods well known inthe art, such as ion-exchange chromatography, or selectiveprecipitation.

It is to be understood that the dyes of the invention have been drawn inone or another particular electronic resonance structure. Every aspectof the instant invention applies equally to dyes that are formally drawnwith other permitted resonance structures, as the electronic charge onthe subject dyes are delocalized throughout the dye itself.

TABLE 1 Spectral properties of selected dyes of the invention ExcitationEmission Quantum Yield† Cpd No. (nm, MeOH) (nm, MeOH) (MeOH) 63 665 6940.55 64 653 695 0.15 65 664 693 0.4 66 686 706 0.35 68 664 696 0.4 70570 592 0.44 72 663 694 0.42 73 664 697 0.48 76 685 705 0.46 81 750 8000.12 †Relative to nile blue in ethanol and CY5 dye in methanolConjugates of Reactive Dyes

In one embodiment of the invention, the dye contains at least one group-L-R_(x), where R_(x) is the reactive group that is attached to the dyeby a covalent linkage L. In certain embodiments, the covalent linkageattaching the dye to R_(x) contains multiple intervening atoms thatserve as a spacer. The dyes with a reactive group (R_(x)) label a widevariety of organic or inorganic substances that contain or are modifiedto contain functional groups with suitable reactivity, resulting inchemical attachment of the conjugated substance (S_(c)), represented by-L-S_(c). As used herein, “reactive group” means moiety on the compoundthat is capable of chemically reacting with a functional group on adifferent compound to form a covalent linkage. Typically the reactivegroup is an electrophile or nucleophile that can form a covalent linkagethrough exposure to the corresponding functional group that is anucleophile or electrophile, respectively. Alternatively, the reactivegroup is a photoactivatable group, and becomes chemically reactive onlyafter illumination with light of an appropriate wavelength. Typically,the conjugation reaction between the reactive dye and the substance tobe conjugated results in one or more atoms of the reactive group R_(x)to be incorporated into a new linkage L attaching the dye to theconjugated substance S_(c). Selected examples of reactive groups andlinkages are shown in Table 2, where the reaction of an electrophilicgroup and a nucleophilic group yields a covalent linkage.

TABLE 2 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers imido esters amines/anilines amidines isocyanates amines/anilinesureas isocyanates alcohols/phenols urethanes isothiocyanatesamines/anilines thioureas maleimides thiols thioethers phosphoramiditesalcohols phosphite esters silyl halides alcohols silyl ethers sulfonateesters amines/anilines alkyl amines sulfonate esters thiols thioetherssulfonate esters carboxylic acids esters sulfonate esters alcoholsethers sulfonyl halides amines/anilines sulfonamides sulfonyl halidesphenols/alcohols sulfonate esters *Activated esters, as understood inthe art, generally have the formula —COΩ, where Ω is a good leavinggroup (e.g. succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy(—OC₄H₃O₂—SO₃H), -1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group oraryloxy substituted one or more times by electron withdrawingsubstituents such as nitro, fluoro, chloro, cyano, or trifluoromethyl,or combinations thereof, used to form activated aryl esters; or acarboxylic acid activated by a carbodiimide to form an anhydride ormixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b),which may be the same or different, are C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl,or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates

The covalent linkage L binds the reactive group R_(X) or conjugatedsubstance S_(C) to the compound, either directly (L is a single bond) orwith a combination of stable chemical bonds, optionally includingsingle, double, triple or aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds,and nitrogen-platinum bonds. L typically includes ether, thioether,carboxamide, sulfonamide, urea, urethane or hydrazine moieties. In oneembodiment, the covalent linkage incorporates a platinum atom, such asdescribed in U.S. Pat. No. 5,714,327 (incorporated by reference).Preferred L moieties have 1-20 nonhydrogen atoms selected from the groupconsisting of C, N, O, P, and S; and are composed of any combination ofether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Preferably L is acombination of single carbon-carbon bonds and carboxamide or thioetherbonds. The longest linear segment of the linkage L preferably contains4-10 nonhydrogen atoms, including one or two heteroatoms. Examples of Linclude substituted or unsubstituted polymethylene, arylene,alkylarylene, arylenealkyl, or arylthio. In one embodiment, L contains1-6 carbon atoms; in another, L comprises a thioether linkage. In yetanother embodiment, L is or incorporates the formula—(CH₂)_(d)(CONH(CH₂)_(e))_(z)—, (CH₂)_(d)(CONH(CH₂)_(e)NH₂)_(z′)—,—(CH₂)_(d)(CONH(CH₂)_(e)NHCO)—_(z′),

where d is 0-5, e is 1-5 and z′ is 0 or 1. In one embodiment, -L is—(CH₂)_(d)(CONH(CH₂)_(e))_(z′)— and —R_(x) is a carboxylic acid, or asuccinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide. Inanother embodiment, -L is —(CH₂)_(d)(CONH(CH₂)_(e))_(z′)— and S_(c) is aconjugated substance that is an antibody or fragment thereof, afluorescent protein, a lectin, an oligonucleotide, a small-moleculedrug, an NTA, or a tyramide.

Choice of the reactive group used to attach the dye to the substance tobe conjugated typically depends on the functional group on the substanceto be conjugated and the type or length of covalent linkage desired. Thetypes of functional groups typically present on the organic or inorganicsubstances include, but are not limited to, amines, amides, thiols,alcohols, phenols, aldehydes, ketones, phosphates, imidazoles,hydrazines, hydroxylamines, disubstituted amines, halides, epoxides,carboxylate esters, sulfonate esters, purines, pyrimidines, carboxylicacids, olefinic bonds, or a combination of these groups. A single typeof reactive site may be available on the substance (typical forpolysaccharides), or a variety of sites may occur (e.g. amines, thiols,alcohols, phenols), as is typical for proteins. A conjugated substancemay be conjugated to more than one dye, which may be the same ordifferent, or to a substance that is additionally modified by a hapten,such as biotin. Although some selectivity can be obtained by carefulcontrol of the reaction conditions, selectivity of labeling is bestobtained by selection of an appropriate reactive dye.

Typically, R_(X) will react with an amine, a thiol, an alcohol, analdehyde or a ketone. Preferably R_(X) reacts with an amine or a thiolfunctional group. In one embodiment, R_(X) is an acrylamide, a reactiveamine (including a cadaverine or ethylenediamine), an activated ester ofa carboxylic acid (typically a succinimidyl ester of a carboxylic acid),an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, ananhydride, an aniline, an aryl halide, an azide, an aziridine, aboronate, a carboxylic acid, a diazoalkane, a haloacetamide, ahalotriazine, a hydrazine (including hydrazides), an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, areactive platinum complex, a sulfonyl halide, or a thiol group. Inanother embodiment, R_(x) is a reactive group that is an activated esterof a carboxylic acid, an amine, a carboxylic acid, a halotriazine, ahydrazide, a maleimide, a reactive platinum complex. By “reactiveplatinum complex” is particularly meant chemically reactive platinumcomplexes such as described in U.S. Pat. Nos. 5,580,990; 5,714,327;5,985,566 (all incorporated by reference).

Where the reactive group is a photoactivatable group, such as an azide,diazirinyl, azidoaryl, or psoralen derivative, the dye becomeschemically reactive only after illumination with light of an appropriatewavelength.

Where R_(x) is an activated ester of a carboxylic acid, the reactive dyeis particularly useful for preparing dye-conjugates of proteins,nucleotides, oligonucleotides, or haptens. Where R_(x) is a maleimide orhaloacetamide the reactive dye is particularly useful for conjugation tothiol-containing substances. Where R_(x) is a hydrazide, the reactivedye is particularly useful for conjugation to periodate-oxidizedcarbohydrates and glycoproteins, and in addition is an aldehyde-fixablepolar tracer for cell microinjection.

Preferably, R_(X) is a carboxylic acid, a succinimidyl ester of acarboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, amaleimide group, an aliphatic amine, a perfluorobenzamido, anazidoperfluorobenzamido group, or a psoralen. More preferably, R_(X) isa succinimidyl ester of a carboxylic acid, a maleimide, aniodoacetamide, or a reactive platinum complex. In a particularembodiment R_(X) is a reactive platinum complex, or a succinimidyl esterof a carboxylic acid. Where R_(X) is a reactive platinum complex, it istypically a haloplatinate or a platinum nitrate.

Based on the above-mentioned attributes, the appropriate reactive dye ofthe invention is selected for the preparation of the desireddye-conjugate, whose advantageous properties make them useful for a widevariety of applications. When compared to conjugates of widely usedcarbocyanine dyes for which the point of attachment is at the 1-positionof the indolium moiety (e.g. Amersham's Cy Dyes™), the dye-conjugates ofthis invention have demonstrably superior optical properties. See, e.g.Example 73 and FIG. 6 comparing protein conjugates of Cy3 dye andspectrally similar conjugates of Compound 13 of the invention (whereinn=1); Tables 3 and 5 with an extensive comparison of protein conjugatesof Cy5 dye with the spectrally similar conjugates of compound 9 of theinvention (wherein n=2); Examples 68-69, 71-72, and 74; and FIGS. 2-3.See also, Example 76 and FIGS. 8 and 9 comparing dye-fluorophoreconjugates; as well as Table 5, Examples 92-94, and FIGS. 11-13comparing dye-nucleic acid conjugates; and Examples 72 and 77, and FIGS.5 and 10 for flow-cytometric comparison of cell populations labeled withdye-conjugates. The desired dye-conjugate is selected based on theintended application.

Particularly useful dye-conjugates include, among others, conjugateswhere Sc is an antigen, steroid, vitamin, drug, hapten, metabolite,toxin, environmental pollutant, amino acid, peptide, protein, nucleicacid, nucleic acid polymer, carbohydrate, lipid, ion-complexing moiety,stable free radical or glass, plastic or other non-biological polymer.Alternatively, Sc is a cell, cellular system, cellular fragment, orsubcellular particle, e.g. inter alia, a virus particle, bacterialparticle, virus component, biological cell (such as animal cell, plantcell, bacteria, yeast, or protist), or cellular component. Reactive dyestypically label functional groups at the cell surface, in cellmembranes, organelles, or cytoplasm.

Typically Sc is an amino acid, peptide, protein, tyramine,polysaccharide, ion-complexing moiety, nucleoside, nucleotide,oligonucleotide, nucleic acid, hapten, psoralen, drug, hormone, lipid,lipid assembly, polymer, polymeric microparticle, biological cell orvirus. More typically, Sc is a peptide, a protein, a nucleotide, anoligonucleotide, or a nucleic acid. When conjugating dyes of theinvention to such biopolymers, it is possible to incorporate more dyesper molecule to increase the fluorescent signal. For example, it ispossible to incorporate at least four molecules of such dyes permolecule of antibody without loss of total fluorescence, whereasfluorescence of the spectrally comparable Cy5 (wherein n=2) is stronglyquenched when greater than approximately two Cy5 dyes are incorporatedper antibody (Example 33 and 34, Table 3). These results confirmproblems with Cy dye conjugates reported by others, e.g. BIOCONJUGATECHEM. 11, 696 (2000). A comparison of commercially available Cy5conjugates or conjugates of known dyes, and the optimally labeledconjugates of the invention are typically at least two-fold, usuallymore than three-fold and sometimes more than four-fold more fluorescentthan conjugates of the Cy5 dye at the same antibody concentration (Table3; FIGS. 2 and 5).

TABLE 3 Protein Source Compound DOS‡ RQY† TF§ GAR IgG Molecular Probes 9 2.10 1.6 3.3 GAR IgG Molecular Probes  9 3.0 1.4 4.3 GAR IgGMolecular Probes  9 4.1 1.1 4.5 GAR IgG Molecular Probes  9 5.2 0.9 4.7GAR IgG Molecular Probes  9 7.6 0.6 4.6 GAR IgG Molecular Probes  9 8.20.5 4.1 GAR IgG Molecular Probes Cy5 2.4 0.95 2.3 GAR IgG MolecularProbes Cy5 4.1 0.5 2.1 GAR IgG Molecular Probes Cy5 4.5 0.2 0.9 GAR IgGMolecular Probes Cy5 5 0.3 1.5 GAR IgG Molecular Probes Cy5 5.8 0.03 0.2GAR IgG Jackson Labs Cy5 2.2 1.1 2.4 GAR IgG Chemicon Cy5 3.3 0.82 2.7GAR IgG Zymed Cy5 4.7 0.53 2.5 GAR IgG Amersham-Pharmacia Biotech Cy55.7 0.22 1.3 GAR IgG Kirkegaard & Perry Cy5 5.7 0.20 1.1 GAR IgGRockland Cy5 10.1 0.05 0.5 GAR IgG Molecular Probes 30 2.7 0.7 1.9 GARIgG Molecular Probes 30 4.3 0.28 1.2 GAR IgG Molecular Probes 30 5.40.10 0.5 GAR IgG Molecular Probes 24 2.9 0.8 2.2 GAR IgG MolecularProbes 24 4.3 0.33 1.4 GAR IgG Molecular Probes 24 5.6 0.15 0.8 GAR IgGMolecular Probes 25 2.0 0.7 1.4 GAR IgG Molecular Probes 25 2.9 0.35 1.0GAR IgG Molecular Probes 25 3.9 0.13 0.5 GAR IgG Molecular Probes 27 2.00.9 1.8 GAR IgG Molecular Probes 27 3.2 0.52 1.6 GAR IgG MolecularProbes 27 4.2 0.28 1.2 GAR IgG Molecular Probes 26 1.4 0.58 0.81 GAR IgGMolecular Probes 26 2.1 0.3 0.61 GAR IgG Molecular Probes 26 2.9 0.10.29 GAM IgG Molecular Probes  9 2.1 1.4 2.9 GAM IgG Molecular Probes  92.2 1.4 3.1 GAM IgG Molecular Probes  9 3.1 1.1 3.5 GAM IgG MolecularProbes  9 4.2 1.2 5.0 GAM IgG Molecular Probes  9 5.2 0.6 3.2 GAM IgGJackson Labs Cy5 1.9 1.0 1.9 GAM IgG Molecular Probes Cy5 2 0.9 1.8 GAMIgG Molecular Probes Cy5 3.3 0.5 1.6 GAM IgG Molecular Probes Cy5 4.80.09 0.4 Concanavalin A Molecular Probes  9 1.7 1.2 2.0 Concanavalin AMolecular Probes  9 2.2 0.8 1.8 Concanavalin A Molecular Probes  9 3.30.9 2.9 Concanavalin A Molecular Probes  9 3.7 0.6 2.3 Concanavalin AMolecular Probes  9 5.5 0.6 3.3 Concanavalin A Molecular Probes  9 5.60.8 4.5 Concanavalin A Molecular Probes Cy5 1.3 0.9 1.2 Concanavalin AMolecular Probes Cy5 2.4 0.5 1.2 Concanavalin A Molecular Probes Cy5 3.10.2 0.6 Concanavalin A Molecular Probes Cy5 3.3 0.8 2.6 Concanavalin AMolecular Probes Cy5 4.4 0.3 1.3 Concanavalin A Molecular Probes Cy5 6.50.1 0.7 Streptavidin Molecular Probes  9 2.2 2.4 5.4 StreptavidinMolecular Probes  9 2.8 1.9 5.3 Streptavidin Molecular Probes  9 3.4 1.96.5 Streptavidin Molecular Probes  9 4.1 1.8 7.3 Streptavidin MolecularProbes  9 4.5 1.7 7.6 Streptavidin Molecular Probes  9 5.2 1.6 8.3Streptavidin Molecular Probes Cy5 1.6 1.8 2.8 Streptavidin MolecularProbes Cy5 2.7 1.5 4.1 Streptavidin Jackson Labs Cy5 3.3 1.8 6.0Streptavidin Amersham-Pharmacia Biotech Cy5 3.6 1.4 5.0 StreptavidinMolecular Probes Cy5 3.6 1.4 5.0 Transferrin Molecular Probes  9 1.8 1.52.7 Transferrin Molecular Probes  9 2.7 1.3 3.5 Transferrin MolecularProbes  9 3.7 1.2 4.4 Transferrin Molecular Probes  9 4.2 1.1 4.4Transferrin Molecular Probes  9 5.8 0.7 4.1 Transferrin Molecular Probes 9 5.9 0.6 3.5 Transferrin Molecular Probes Cy5 0.8 1.1 0.9 TransferrinMolecular Probes Cy5 1.4 0.8 1.1 Transferrin Molecular Probes Cy5 2.70.5 1.4 Transferrin Molecular Probes Cy5 3.0 0.7 2.1 TransferrinMolecular Probes Cy5 5.3 0.1 0.5 Transferrin Molecular Probes Cy5 5.80.02 0.1 †RQY (Relative Quantum Yield) is measured by matchingabsorbance between bioconjugate and DDAO(7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridine-2-one)), and comparingintegrated fluorescence emission intensities. ‡DOS (Degree ofSubstitution) is the approximate molar ratio of the dye to proteinfollowing conjugation. DOS estimated using Molar Extinction coefficientsof 250,000 M⁻¹ cm⁻¹ for Cy5 and 239,000 M⁻¹ cm⁻¹ for Compound 9bioconjugates respectively, and protein extinction coefficients from theliterature and a visible dye-correction term (at 280 nm) of 3-5%. §TF(Total Fluorescence) is proportional to the overall brightness of thebioconjugate, and is defined as the product of the RQY and DOS: TF = RQY× DOS.

Alternatively, Sc is a ligand or a hapten, such as biotin. A preferredconjugate is a phenol such as a tyramine (e.g. as described in U.S. Pat.Nos. 5,196,306; 5,583,001; 5,731,158; all incorporated by reference),wherein the conjugate is useful as a substrate for horseradishperoxidase (Example 82).

In one embodiment, Sc is a biological polymer such as a peptide,protein, oligonucleotide, or nucleic acid polymer that is also labeledwith at least a second non-fluorescent or fluorescent dye (optionally anadditional dye of the present invention), to form an energy-transferpair. In some aspects of the invention, the labeled conjugate functionsas an enzyme substrate, and enzymatic hydrolysis disrupts the energytransfer. Alternatively, Sc is itself a fluorescent or nonfluorescentdye, optionally an additional dye of the present invention, whichdye-conjugate forms a labeling complex that exhibits a large Stokesshift due to internal energy-transfer (as described in U.S. Pat. No.6,008,373 above), which complex is useful to label an organic orinorganic substance (Examples 76-77; FIGS. 8-10).

In one embodiment, S_(C) is an amino acid (including those that areprotected or are substituted by phosphates, carbohydrates, or C₁ to C₂₂carboxylic acids), or is a polymer of amino acids such as a peptide orprotein. Preferred conjugates of peptides contain at least five aminoacids, more preferably 5 to 36 amino acids. Preferred peptides include,but are not limited to, neuropeptides, cytokines, toxins, proteasesubstrates, and protein kinase substrates. Preferred protein conjugatesinclude enzymes, antibodies, lectins, glycoproteins, histones, albumins,lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins,chemokines and growth factors. In one preferred aspect, the conjugatedprotein is a phycobiliprotein, such as allophycocyanin, phycocyanin,phycoerythrin, allophycocyanin B, B-phycoerythrin, phycoerythrocyanin,and b-phycoerythrin (for example, see U.S. Pat. No. 5,714,386 toRoederer (1998), incorporated by reference). Particularly preferred areconjugates of R-phycoerythrin and of allophycocyanin with selected dyesof the invention that serve as excited-state energy acceptors or donors.In these conjugates, excited state energy transfer results in longwavelength fluorescence emission when excited at relatively shortwavelengths (Example 76, FIGS. 8 and 10). In another aspect of theinvention, the conjugated protein is an antibody, an antibody fragment,avidin, streptavidin, a toxin, a lectin, a hormone, a chemokine, or agrowth factor. Typically, where the conjugated substance is a toxin, itis a neuropeptide or a phallotoxin, such as phalloidin (Example 64).

In another embodiment, S_(C) is a nucleic acid base, nucleoside,nucleotide (Example 87) or a nucleic acid polymer (Examples 88-93),including those that are modified to possess an additional linker orspacer for attachment of the dyes of the invention, such as an alkynyllinkage (U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S. Pat. No.4,711,955), or a heteroatom-substituted linker (U.S. Pat. No. 5,684,142)(all patents incorporated by reference), or other linkage. In anotherembodiment, the conjugated substance is a nucleoside or nucleotideanalog that links a purine or pyrimidine base to a phosphate orpolyphosphate moiety through a noncyclic spacer. In a third embodiment,the dye is conjugated to the carbohydrate portion of a nucleotide ornucleoside, typically through a hydroxyl group but additionally througha thiol or amino group (U.S. Pat. No. 5,659,025, U.S. Pat. No.5,668,268, U.S. Pat. No. 5,679,785; all incorporated by reference).Typically, the conjugated nucleotide is a nucleoside triphosphate or adeoxynucleoside triphosphate or a dideoxynucleoside triphosphate.Typically, the nucleotide is a cytidine or uridine or deoxy or dideoxycytidine or uridine. Incorporation of methylene moieties or nitrogen orsulfur heteroatoms into the phosphate or polyphosphate moiety is alsouseful. Nonpurine and nonpyrimidine bases such as 7-deazapurines (U.S.Pat. No. 6,150,510, incorporate by reference) and nucleic acidscontaining such bases can also be coupled to dyes of the invention.Nucleic acid adducts prepared by reaction of depurinated nucleic acidswith amine, hydrazide or hydroxylamine derivatives provide an additionalmeans of labeling and detecting nucleic acids, e.g. “A method fordetecting abasic sites in living cells: age-dependent changes in baseexcision repair.” Atamna H, Cheung I, Ames B N. Proc Natl Acad Sci USA97, 686-691 (2000); incorporated by reference.

Preferred nucleic acid polymer conjugates are labeled, single- ormulti-stranded, natural or synthetic DNA or RNA, DNA or RNAoligonucleotides, or DNA/RNA hybrids, or incorporate an unusual linkersuch as morpholine derivatized phosphates (AntiVirals, Inc., CorvallisOreg.), or peptide nucleic acids such as N-(2-aminoethyl)glycine units.When the nucleic acid is a synthetic oligonucleotide, it typicallycontains fewer than 50 nucleotides, more typically fewer than 25nucleotides. Conjugates of peptide nucleic acids (PNA) (Nielsen et alU.S. Pat. No. 5,539,082, incorporated by reference) may be preferred forsome applications because of their generally faster hybridization rates.

Fluorescent nucleic acid polymers are typically prepared from labelednucleotides or oligonucleotides using oligonucleotide-primed DNApolymerization (Example 93), such as by using the polymerase chainreaction or through primer extension, or by terminal-transferasecatalyzed addition of a labeled nucleotide to a 3′-end of a nucleic acidpolymer. Fluorescent RNA polymers are typically prepared from labelednucleotides by transcription. Typically, the dye is attached via one ormore purine or pyrimidine bases through an amide, ester, ether orthioether bond; or is attached to the phosphate or carbohydrate by abond that is an ester, thioester, amide, ether or thioether.Alternatively, dye conjugate of the invention is simultaneously labeledwith a hapten such as biotin or digoxigenin, or to an enzyme such asalkaline phosphatase, or to a protein such as an antibody. Nucleotideconjugates of the invention are readily incorporated by DNA polymeraseand can be used for in situ hybridization and nucleic acid sequencing(e.g., U.S. Pat. Nos. 5,332,666; 5,171,534; and 4,997,928, and WO Appl.94/05688; all incorporated by reference). In another aspect of theinvention, the oligonucleotide incorporates an aliphatic amine, which issubsequently conjugated to an amine-reactive dye of the invention or athiol or thiophosphate, which is conjugated to a thiol-reactive dye ofthe invention. In yet another aspect of the invention, the purine basesof the oligonucleotide react with a reactive metal complex (preferably aplatinum complex) bound to a dye of the invention, yielding adye-conjugate (Example 92). Nucleic acid conjugates of dyes of theinvention that are linked at the 3-position of the indolium ringunexpectedly have spectral properties that are superior to those ofstructurally similar carbocyanine dyes wherein the dye is not linked atthe 3-position of the indolium ring (Examples 92-94, FIGS. 11-12, Table8).

In one embodiment, the conjugated oligonucleotides of the invention areaptamers for a particular target molecule, such as a metabolite, dye,hapten, or protein. That is, the oligonucleotides have been selected tobind preferentially to the target molecule. Methods of preparing andscreening aptamers for a given target molecule have been previouslydescribed and are known in the art (for example, U.S. Pat. No. 5,567,588to Gold (1996), incorporated by reference).

In another embodiment, the conjugated substance (S_(c)) is acarbohydrate that is typically a polysaccharide, such as a dextran,FICOLL™, heparin, glycogen, amylopectin, mannan, inulin, starch, agaroseand cellulose. Alternatively, the carbohydrate is a polysaccharide thatis a lipopolysaccharide. Preferred polysaccharide conjugates aredextran, FICOLL™, or lipopolysaccharide conjugates.

In another embodiment, the conjugated substance (S_(c)), is a lipid(typically having 6-60 carbons), including glycolipids, phospholipids,sphingolipids, and steroids. Alternatively, the conjugated substance isa lipid assembly, such as a liposome. The lipophilic moiety may be usedto retain the conjugated substances in cells, as described in U.S. Pat.No. 5,208,148 (incorporated by reference). Certain polar dyes of theinvention may also be trapped within lipid assemblies.

Conjugates having an ion-complexing moiety serve as indicators forcalcium, sodium, magnesium, zinc, potassium, or other biologicallyimportant metal ions. Preferred ion-complexing moieties are crown ethers(U.S. Pat. No. 5,405,975 and provisional patent application titled“Crown Ether Derivatives”, filed Dec. 19, 2000 by Martin et al. U.S.Ser. No. 60/258,266, both incorporated by reference); derivatives of1,2-bis-(2-aminophenoxyethane)-N,N,N′,N′-tetraacetic acid (BAPTAchelators; U.S. Pat. No. 5,453,517, U.S. Pat. No. 5,516,911, and U.S.Pat. No. 5,049,673, all incorporated by reference); derivatives of2-carboxymethoxyaniline-N,N-diacetic acid (APTRA chelators; AM. J.PHYSIOL. 256, C540 (1989), incorporated by reference); or pyridine- andphenanthroline-based metal ion chelators (U.S. Pat. No. 5,648,270,incorporated by reference); or derivatives of nitrilotriacetic acid(NTA), see e.g. “Single-step synthesis and characterization ofbiotinylated nitrilotriacetic acid, a unique reagent for the detectionof histidine-tagged proteins immobilized on nitrocellulose”, McMahan SA, Burgess R R. Anal Biochem 236, 101-106 (1996); incorporated byreference. Preferably, the ion-complexing moiety is a crown etherchelator, a BAPTA chelator, an APTRA chelator or a derivative ofnitrilotriacetic acid.

Other conjugates of non-biological materials include dye-conjugates oforganic or inorganic polymers, polymeric films, polymeric wafers,polymeric membranes, polymeric particles, or polymeric microparticles(Example 84); including magnetic and non-magnetic microspheres; iron,gold or silver particles; conducting and non-conducting metals andnon-metals; and glass and plastic surfaces and particles. Conjugates areoptionally prepared by copolymerization of a dye that contains anappropriate functionality while preparing the polymer, or by chemicalmodification of a polymer that contains functional groups with suitablechemical reactivity. Other types of reactions that are useful forpreparing dye-conjugates of polymers include catalyzed polymerizationsor copolymerizations of alkenes and reactions of dienes withdienophiles, transesterifications or transaminations. In anotherembodiment, the conjugated substance is a glass or silica, which may beformed into an optical fiber or other structure.

In one aspect of the invention, S_(c) is a conjugated substance that isan antibody (including intact antibodies, antibody fragments, andantibody sera, etc.), an amino acid, blood vessel proliferationinhibition factors (including Angiostatin™, Endostatin™, etc.), anavidin or streptavidin, a biotin (e.g. an amidobiotin, a biocytin, adesthiobiotin, etc.), a blood component protein (e.g. an albumin, afibrinogen, a plasminogen, etc.), a dextran, an enzyme, an enzymeinhibitor, an IgG-binding protein (e.g. a protein A, protein G, proteinA/G, etc.), a fluorescent protein (e.g. a phycobiliprotein, an aequorin,a green fluorescent protein, etc.), a growth factor, a hormone, a lectin(e.g. a wheat germ agglutinin, a conconavalin A, etc.), alipopolysaccharide, a metal-binding protein (e.g. a calmodulin, etc.), amicroorganism or portion thereof (e.g. a bacteria, a virus, a yeast,etc.), a neuropeptide and other biologically active factors (e.g. adermorphin, a deltropin, an endomorphin, an endorphin, a tumor necrosisfactor etc.), a non-biological microparticle (e.g. of ferrofluid, gold,polystyrene, etc.), a nucleotide, an oligonucleotide, a peptide toxin(e.g. an apamin, a bungarotoxin, a phalloidin, etc.), aphospholipid-binding protein (e.g. an annexin, etc.), a small-moleculedrug (e.g. a methotrexate, etc.), a structural protein (e.g. an actin, afibronectin, a laminin, a microtubule-associated protein, a tublin,etc.), an NTA, or a tyramide. Typically, S_(c) is a conjugated substancethat is an antibody, an amino acid, an avidin or streptavidin, a biotin,blood vessel proliferation inhibition factor, a blood component protein,a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG-bindingprotein, a fluorescent protein, a growth factor, a lectin, alipopolysaccharide, a metal-binding protein, a microorganism or portionthereof, a neuropeptide, a non-biological microparticle, a nucleotide,an oligonucleotide, a peptide toxin, a phospholipid-binding protein, asmall-molecule drug, a structural protein, an NTA, or a tyramidePreferably, S_(c) is a conjugated substance that is an actin, anantibody or fragment thereof, an avidin or streptavidin, a biotin, adextran, an enzyme, a fluorescent protein, a lectin, alipopolysaccharide, a microorganism, a non-biological microparticle, anucleotide, an oligonucleotide, a peptide toxin, aphosphotidylserine-binding protein, a protein A or G, a small-moleculedrug, an NTA, or a tyramide. In another embodiment, where R³ is areactive group, another substituent contains a conjugated substanceS_(c) that is an amino acid, peptide, polypeptide, protein, nucleotide,oligonucleotide, nucleic acid polymer, a sugar, a polysaccharide, anoligosaccharide, a fluorescent dye, or a microsphere.

In one embodiment, conjugates of biological polymers such as peptides,proteins, oligonucleotides, nucleic acid polymers are also labeled withat least a second fluorescent or nonfluorescent dye, that is optionallyan additional dye of the present invention, to form an energy-transferpair. In some aspects of the invention, the labeled conjugate functionsas an enzyme substrate, and enzymatic hydrolysis disrupts the energytransfer. Alternatively, the conjugated substance is itself afluorescent or nonfluorescent dye, optionally an additional dye of thepresent invention, that forms a labeling complex that exhibits a largeStokes shift due to internal energy-transfer (as described in U.S. Pat.No. 6,008,373 to Waggoner et al., (1999), incorporated by reference). Inanother embodiment of the invention, the energy-transfer pair thatincorporates a dye of the invention is conjugated to an oligonucleotidethat displays efficient fluorescence quenching in its hairpinconformation (the so-called “molecular beacons” of Tyagi et al., NATUREBIOTECHNOLOGY 16, 49 (1998) incorporated by reference) or fluorescenceenergy transfer.

The preparation of dye conjugates using reactive dyes is welldocumented, e.g. by R. Haugland, MOLECULAR PROBES HANDBOOK OFFLUORESCENT PROBES AND RESEARCH CHEMICALS, Chapters 1-3 (1996); andBrinkley, BIOCONJUGATE CHEM., 3, 2 (1992). Conjugates typically resultfrom mixing appropriate reactive dyes and the substance to be conjugatedin a suitable solvent in which both are soluble. The majority of thedyes of the invention are readily soluble in aqueous solutions,facilitating conjugation reactions with most biological materials. Forthose reactive dyes that are photoactivated, conjugation requiresillumination of the reaction mixture to activate the reactive dye.

Labeled members of a specific binding pair are typically used asfluorescent probes for the complementary member of that specific bindingpair, each specific binding pair member having an area on the surface orin a cavity that specifically binds to and is complementary with aparticular spatial and polar organization of the other. Preferredspecific binding pair members are proteins that bind non-covalently tolow molecular weight ligands, such as biotin, drug-haptens andfluorescent dyes (such as an anti-fluorescein antibody). Representativespecific binding pairs are shown in Table 4.

TABLE 4 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor toxin toxin receptor carbohydrate lectin or carbohydratereceptor peptide peptide receptor protein protein receptor enzymesubstrate enzyme DNA (RNA) aDNA (aRNA)† hormone hormone receptor ionchelator psoralen nucleic acid target molecule RNA or DNA aptamer *IgGis an immunoglobulin †aDNA and aRNA are the antisense (complementary)strands used for hybridizationSynthesis

Synthesis of the carbocyanine dyes of the invention, where attachment isat the 3-position of the indolium, depends on initial preparation ofcertain key intermediates. The intermediates have the following generalstructure (for simplicity, all but a few of the possible substituentsare shown as hydrogen):

These basic structures are optionally further substituted, during orafter synthesis, to give the corresponding dye substituents as definedabove.

The novel key intermediates are readily synthesized by a reaction thatis analogous to a Fischer indole synthesis (where X is a desiredsubstituent on the resulting indolium, typically sulfo, and R³ and R⁴are as defined above):

In this reaction, an appropriately substituted aryl hydrazine, which istypically a phenylhydrazine of an appropriately substituted naphthylhydrazine, is reacted with an appropriately substituted methyl ketone toyield a 3,3-disubstituted 2-methylindoline derivative. One of the3-position substituents is selected to be a chemically reactive moietyor a group that is converted to a chemically reactive moiety such as acarboxylic acid derivative (Examples 1, 2, 7), an alcohol (Example 16)or an amine (Example 17). It is particularly suitable to utilize asulfonated phenylhydrazine derivative (as in Examples 1-3) or asulfonated naphthylhydrazine derivative (as in Example 25) to increasethe solubility of the final dye. The 3,3-disubstituted-2-methylindole isthen quaternized on the nitrogen atom to an indolium derivative with analkylating agent that is typically an alkyl halide such as ethyl iodide,an alkylsulfonate such as methyl p-toluenesulfonate (Example 7) or acyclic sulfonate such as propanesultone or butanesultone (Examples 2-3).Typically, the key indolium or benzoindolium intermediates aresulfonated one or more times before or after quaternization at R² andsubsequent condensation with the benzazolium moiety and polymethinemoiety to form the subject dyes. Methods for synthesis of dyes whereinn=1, n=2 and n=3 are provided in Examples 12, 8, and 21, respectively.Variations on these methods are well known in the art that yieldsubstituents on the polymethine bridge or on the indolium or benzoliumportion of the dye precursor.

In one embodiment, the synthetic intermediate is a compound of theformula:

or its salts, wherein R³ is C₃-C₇ carboxyalkyl; and R⁴ is a C₁-C₆ alkyl;R² is H or a C₁-C₆ alkyl or C₁-C₆ alkyl substituted by sulfo; R⁶ throughR⁹ are independently H, amino, sulfo, trifluoromethyl, or halogen; orC₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, eachof which is optionally further substituted by carboxy, sulfo, amino, orhydroxy; or any two adjacent substituents of R⁵ through R⁹ form a fusedbenzo ring that is optionally substituted one or more times by amino,sulfo, trifluoromethyl, or halogen; or by C₁-C₆ alkyl, C₁-C₆ alkoxy,C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each of which is optionallyfurther substituted by carboxy, sulfo, amino, or hydroxy. In a preferredembodiment, R³ is C₅-C₆ carboxyalkyl; and R⁴ is methyl; R² is methyl orsulfopropyl; and R⁶ and R⁷ form a fused benzo ring that is optionallysubstituted one or more times by sulfo; or R⁷ is independently H orsulfo, and R⁶ is H; both R³ and R⁹ are H.

A useful synthetic route to the azacarbocyanine dyes of the presentinvention can be described in three parts, following the naturalbreakdown in the description of the compounds. In general, the synthesisof these dyes requires three precursors: the appropriate benzazolium orazabenzazolium salt (the “A” and “B” moieties), and a source for thepolymethine spacer. Typically each component is selected so as toincorporate the appropriate chemical substituents, or functional groupsthat can be converted to the appropriate substituents. The chemistrythat is required to prepare and combine these precursors so as to yieldany of the subject derivatives is generally well understood by oneskilled in the art. Although there are many possible variations that mayyield an equivalent result, we provide herein some useful generalmethods for their synthesis and incorporation of chemical modifications.

Although the chemically reactive azabenzazolium dyes and theirconjugates described herein have not previously been described, avariety of nonreactive azabenzazolium derivatives have been previouslydescribed (see, for example, Brooker, et al., J. AM. CHEM. SOC., 64, 199(1942); Heravi, et al., INDIAN J. CHEM. 36B, 1025 (1997); Smith et al.SULFUR LETTERS 17, 197 (1994); Chu-Moyer et al. J. ORG. CHEM. 60, 5721(1995); Turner, J. ORG. CHEM. 48, 3401 (1983); Couture et al. J.HETEROCYCLIC CHEM. 24, 1765 (1987); Petric et al. J. HETEROCYCLIC CHEM.14, 1045, (1977); Barlin et al. AUST. J. CHEM., 37, 1729 (1984);Saikachi et al. CHEM. & PHARM. BULL. 9, 941 (1961); Barlin AUST. J.CHEM. 36, 983 (1983); Foye et al., J. PHARM. SCI. 64, 1371 (1975);Khanna et al. J. ORG. CHEM. 60, 960 (1995)); British Patent No. 870,753to Ficken et al. (1961); Ficken et al., “Diazaindenes and TheirQuaternary Salts—Part I” pp 3202-3212 (1959); Ficken et al.,“Diazaindenes and Their Quaternary Salts—Part II” pp 584-588 (1961).Synthetic methods for preparing some azabenzazolium precursors have alsobeen described in copending application Ser. No. 09/557,275 by Hauglandet al., filed Apr. 24, 2000, hereby incorporated by reference.

The substituents on the aromatic carbons of the azabenzazolium moietyare typically incorporated in the parent aza- or polyazabenzazolemolecule prior to quaternization with an alkylating agent. However, suchsubstituents may also be incorporated during the synthesis of theazabenzazole moiety. R²/R¹² is usually obtained by alkylation of theparent heterocycle with an alkylating agent that incorporates thedesired R²/R¹² moiety.

The B moiety intermediate is optionally an azabenzazolium precursor, asdescribed above, or is a benzazolium precursor, as well known in the art(for example, U.S. Pat. No. 5,436,134 to Haugland et al., (1995),incorporated by reference). The B moiety is optionally fused toadditional rings, resulting in dyes that absorb and emit at longerwavelengths (for example, see U.S. Pat. No. 6,027,709 to Little et al.(2000), incorporated by reference).

Alkyl, alkoxy, carboxyl, and halogen substituents at aromatic carbonsare typically already present as substituents on the benzazole orazabenzazole precursors, or on compounds that are readily converted tosuch precursors using methods well-known in the art. Sulfonic acidgroups are typically introduced on the precursors prior to condensationof the cyanine dye (for example, see U.S. Pat. No. 5,767,287 to Bobrowet al. (1998), incorporated by reference). Aminoalkyl groups aretypically substituted by a protecting group when they are firstintroduced, typically by substitution onto the benzazole or azabenzazoleprecursor. The protecting group is then removed after condensation ofthe cyanine dye. Aromatic amino groups are typically prepared via thereduction of a nitro substituted benzazolium precursor, which in turn isprepared by the nitration of the benzazole precursor.

The BRIDGE moiety typically originates from the coupling agent used inthe dye construction. For example, N,N′-diphenylformamidine andtriethylorthoformate yields BRIDGE moieties wherein a and b are bothzero. Malonaldehyde bis(phenyl)mine) hydrochloride,1,1,3-trimethoxypropane, and 1,1,3,3-tetramethoxypropane yield dyeswherein one of a and b is 1, and glutaconaldehyde dianil monochlorideyields dyes wherein both a and b are 1.

The methods for synthesis of dyes that contain a variety of reactivegroups such as those described in Table 2 are well documented in theart. Particularly useful are amine-reactive dyes such as “activatedesters” of carboxylic acids, which are typically synthesized by couplinga carboxylic acid to a relatively acidic “leaving group”. Otherpreferred amine-reactive groups include sulfonyl halides, which areprepared from sulfonic acids using a halogenating agent such as PCl₅ orPOCl₃; halotriazines, which are prepared by the reaction of cyanurichalides with amines; and isothiocyanates or isothiocyanates, which areprepared from amines and phosgene or thiophosgene, respectively.

Dyes containing amines and hydrazides are particularly useful forconjugation to carboxylic acids, aldehydes and ketones. Most often theseare synthesized by reaction of an activated ester of a carboxylic acidor a sulfonyl halide with a diamine, such as cadaverine, or with ahydrazine. Alternatively, aromatic amines are commonly synthesized bychemical reduction of a nitroaromatic compound. Amines and hydrazinesare particularly useful precursors for synthesis of thiol-reactivehaloacetamides or maleimides by standard methods.

Nucleosides and nucleotides labeled with dyes of the invention areparticularly useful for some applications of nucleic acid labeling. Theuse of carbocyanine-amidites for labeling nucleotides and nucleosideshave been previously described (U.S. Pat. No. 5,986,086 to Bruch et al.(1999); U.S. Pat. No. 5,808,044 to Brush et al. (1998); U.S. Pat. No.5,556,959 to Brush et al. (1996); all incorporated by reference).

Examples of some synthetic strategies for selected dyes of theinvention, as well as their characterization, synthetic precursors,conjugates and method of use are provided in the examples below. Furthermodifications and permutations will be obvious to one skilled in theart.

Applications and Methods of Use

In one aspect of the invention, the dye compounds of the invention areused to directly stain or label a sample so that the sample can beidentified or quantitated. For instance, such dyes may be added as partof an assay for a biological target analyte, as a detectable tracerelement in a biological or non-biological fluid; or for such purposes asphotodynamic therapy of tumors, in which a dyed sample is irradiated toselectively destroy tumor cells and tissues; or to photoablate arterialplaque or cells, usually through the photosensitized production ofsinglet oxygen. In one preferred embodiment, the dye conjugate is usedto stain a sample that comprises a ligand for which the conjugatedsubstance is a complementary member of a specific binding pair (e.g.Table 4).

Typically, the sample is obtained directly from a liquid source or as awash from a solid material (organic or inorganic) or a growth medium inwhich cells have been introduced for culturing, or a buffer solution inwhich cells have been placed for evaluation. Where the sample comprisescells, the cells are optionally single cells, including microorganisms,or multiple cells associated with other cells in two or threedimensional layers, including multicellular organisms, embryos, tissues,biopsies, filaments, biofilms, etc.

Alternatively, the sample is a solid, optionally a smear or scrape or aretentate removed from a liquid or vapor by filtration. In one aspect ofthe invention, the sample is obtained from a biological fluid, includingseparated or unfiltered biological fluids such as urine, cerebrospinalfluid, blood, lymph fluids, tissue homogenate, interstitial fluid, cellextracts, mucus, saliva, sputum, stool, physiological secretions orother similar fluids. Alternatively, the sample is obtained from anenvironmental source such as soil, water, or air; or from an industrialsource such as taken from a waste stream, a water source, a supply line,or a production lot.

In yet another embodiment, the sample is present on or in solid orsemi-solid matrix. In one aspect of the invention, the matrix is amembrane. In another aspect, the matrix is an electrophoretic gel, suchas is used for separating and characterizing nucleic acids or proteins,or is a blot prepared by transfer from an electrophoretic gel to amembrane. In another aspect, the matrix is a silicon chip or glassslide, and the analyte of interest has been immobilized on the chip orslide in an array (e.g. the sample comprises proteins or nucleic acidpolymers in a microarray). In yet another aspect, the matrix is amicrowell plate or microfluidic chip, and the sample is analyzed byautomated methods, typically by various methods of high-throughputscreening, such as drug screening.

The dye compounds of the invention are generally utilized by combining adye compound of the invention as described above with the sample ofinterest under conditions selected to yield a detectable opticalresponse. The term “dye compound” is used herein to refer to all aspectsof the claimed dyes, including both reactive dyes and dye conjugates.The dye compound typically forms a covalent or non-covalent associationor complex with an element of the sample, or is simply present withinthe bounds of the sample or portion of the sample. The sample is thenilluminated at a wavelength selected to elicit the optical response.Typically, staining the sample is used to determine a specifiedcharacteristic of the sample by further comparing the optical responsewith a standard or expected response.

A detectable optical response means a change in, or occurrence of, anoptical signal that is detectable either by observation orinstrumentally. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of staining, compared with a standard or expected response,indicates whether and to what degree the sample possesses a givencharacteristic. Some dyes of the invention may exhibit littlefluorescence emission, but are still useful as chromophoric dyes. Suchchromophores are useful as energy acceptors in FRET applications, or tosimply impart the desired color to a sample or portion of a sample.

For biological applications, the dye compounds of the invention aretypically used in an aqueous, mostly aqueous or aqueous-misciblesolution prepared according to methods generally known in the art. Theexact concentration of dye compound is dependent upon the experimentalconditions and the desired results, but typically ranges from about onenanomolar to one millimolar or more. The optimal concentration isdetermined by systematic variation until satisfactory results withminimal background fluorescence are accomplished.

The dye compounds are most advantageously used to stain samples withbiological components. The sample may comprise heterogeneous mixtures ofcomponents (including intact cells, cell extracts, bacteria, viruses,organelles, and mixtures thereof), or a single component or homogeneousgroup of components (e.g. natural or synthetic amino acid, nucleic acidor carbohydrate polymers, or lipid membrane complexes). These dyes aregenerally non-toxic to living cells and other biological components,within the concentrations of use.

The dye compound is combined with the sample in any way that facilitatescontact between the dye compound and the sample components of interest.Typically, the dye compound or a solution containing the dye compound issimply added to the sample. Certain dyes of the invention, particularlythose that are substituted by one or more sulfonic acid moieties, tendto be impermeant to membranes of biological cells, and once insideviable cells are typically well retained. Treatments that permeabilizethe plasma membrane, such as electroporation, shock treatments or highextracellular ATP can be used to introduce selected dye compounds intocells. Alternatively, selected dye compounds can be physically insertedinto cells, e.g. by pressure microinjection, scrape loading, patch clampmethods, or phagocytosis.

Dyes that incorporate an aliphatic amine or a hydrazine residue can bemicroinjected into cells, where they can be fixed in place by aldehydefixatives such as formaldehyde or glutaraldehyde. This fixability makessuch dyes useful for intracellular applications such as neuronaltracing.

Dye compounds that possess a lipophilic substituent, such asphospholipids, will non-covalently incorporate into lipid assemblies,e.g. for use as probes for membrane structure; or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials; or for tracing. Lipophilic dyes are useful asfluorescent probes of membrane structure.

Chemically reactive dye compounds will covalently attach to acorresponding functional group on a wide variety of materials, formingdye conjugates as described above. Using dye compounds to label reactivesites on the surface of cells, in cell membranes or in intracellularcompartments such as organelles, or in the cell's cytoplasm, permits thedetermination of their presence or quantity, accessibility, or theirspatial and temporal distribution in the sample. Photoreactive dyes canbe used similarly to photolabel components of the outer membrane ofbiological cells or as photo-fixable polar tracers for cells.

Preferred compounds used for methods of the invention include thosewhere R_(x) is a carboxylic acid, an activated ester of a carboxylicacid, an amine, an azide, a hydrazide, a haloacetamide, an alkyl halide,an isothiocyanate, or a maleimide group; and those where S_(c) is anantibody, a peptide, a lectin, a polysaccharide, a nucleotide, anucleoside, an oligonucleotide, a nucleic acid polymer, anion-complexing moiety, a lipid, or a non-biological organic polymer orpolymeric microparticle, that is optionally bound to one or moreadditional fluorophores that are the same or different.

Optionally, the sample is washed after staining to remove residual,excess or unbound dye compound. The sample is optionally combined withone or more other solutions in the course of staining, including washsolutions, permeabilization and/or fixation solutions, and solutionscontaining additional detection reagents. An additional detectionreagent typically produces a detectable response due to the presence ofa specific cell component, intracellular substance, or cellularcondition, according to methods generally known in the art. Where theadditional detection reagent has, or yields a product with, spectralproperties that differ from those of the subject dye compounds,multi-color applications are possible. This is particularly useful wherethe additional detection reagent is a dye or dye-conjugate of thepresent invention having spectral properties that are detectablydistinct from those of the staining dye.

The compounds of the invention that are dye conjugates are usedaccording to methods extensively known in the art; e.g. use of antibodyconjugates in microscopy and immunofluorescent assays; and nucleotide oroligonucleotide conjugates for nucleic acid hybridization assays andnucleic acid sequencing (e.g., U.S. Pat. Nos. 5,332,666 to Prober, etal. (1994); 5,171,534 to Smith, et al. (1992); 4,997,928 to Hobbs(1991); and WO Appl. 94/05688 to Menchen, et al.; all incorporated byreference). Dye-conjugates of multiple independent dyes of the inventionpossess utility for multi-color applications.

At any time after or during staining, the sample is illuminated with awavelength of light selected to give a detectable optical response, andobserved with a means for detecting the optical response. Equipment thatis useful for illuminating the dye compounds of the invention includes,but is not limited to, hand-held ultraviolet lamps, mercury arc lamps,xenon lamps, lasers and laser diodes. These illumination sources areoptionally integrated into laser scanners, fluorescence microplatereaders, standard or minifluorometers, or chromatographic detectors.Preferred embodiments of the invention are dyes that are be excitable ator near the wavelengths 633-636 nm, 647 nm, 660 nm, 680 nm and beyond700 nm, as these regions closely match the output of relativelyinexpensive excitation sources.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, examination of the sample optionallyincludes sorting portions of the sample according to their fluorescenceresponse.

Kits

One aspect of the instant invention is the formulation of kits thatfacilitate the practice of various assays using any of the dyes of theinvention, as described above. The kits of the invention typicallycomprise a colored or fluorescent dye of the invention, either presentas a chemically reactive label useful for preparing dye-conjugates, orpresent as a dye-conjugate where the conjugated substance is a specificbinding pair member, or a nucleoside, nucleotide, oligonucleotide,nucleic acid polymer, peptide, or protein. The kit optionally furthercomprises one or more buffering agents, typically present as an aqueoussolution. The kits of the invention optionally further compriseadditional detection reagents, a purification medium for purifying theresulting labeled substance, luminescence standards, enzymes, enzymeinhibitors, organic solvent, or instructions for carrying out an assayof the invention.

Preferred kits of the invention include those that include a dyesolution comprising a compound of the formula

or its salts, wherein R³ is -L-R_(x); or -L-S_(c); and R⁴ is C₁-C₆alkyl; and L is a covalent linkage that is linear or branched, cyclic orheterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atomsselected from the group consisting of C, N, P, O, and S, andincorporating the formula —(CH₂)_(d)(CONH(CH₂)_(e))_(z′)—, where d is0-5, e is 1-5 and z′ is 0 or 1, such that the linkage contains anycombination of ether, thioether, amine, ester, amide bonds; or single,double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen,phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, ornitrogen-platinum bonds; or aromatic or heteroaromatic bonds; R_(x) is areactive group that is an activated ester of a carboxylic acid, anamine, a carboxylic acid, a halotriazine, a hydrazide, a maleimide, areactive platinum complex; S_(c) is a conjugated substance that is anantibody, an amino acid, an angiostatin or endostatin, an avidin orstreptavidin, a biotin, a blood component protein, a dextran, an enzyme,an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescentprotein, a growth factor, a lectin, a lipopolysaccharide, ametal-binding protein, a microorganism or portion thereof, aneuropeptide, a non-biological microparticle, a nucleotide, anoligonucleotide, a peptide toxin, a phospholipid-binding protein, asmall-molecule drug, a structural protein, an NTA, or a tyramide; and R⁷is sulfo, and R⁶, R⁸, and R⁹ are H; or R⁶ and R⁷ combine to form a fusedbenzo ring that is substituted one or more times by sulfo, and R³ and R⁹are H; the atoms of W are selected from —CH, —C, —CR^(1′), and—N(R¹²)_(β′), where each β′ is 1, but no more than one of such atoms is—N(R¹²)_(β′), where each R^(1′) is sulfo or Br; R² and R¹² areindependently a C₁-C₆ alkyl or C₁-C₆ alkyl substituted by sulfo; δ is 0or 1, and δ+all δ′=1; and δ is 1 only when W contains no ring nitrogenatoms; R¹³ and R¹⁴ are independently C₁-C₆ alkyl; each of R²¹, R²², andR²³ are H; or any two adjacent substituents of R²¹, R²², R²³, when takenin combination, forms a 4-, 5-, or 6-membered saturated or unsaturatedhydrocarbon ring that is optionally substituted one or more times byC₁-C₆ alkyl, halogen, or a carbonyl oxygen.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention. See also, U.S. Patent application Ser. No. 60/236,637,filed Sep. 29, 2000, incorporated by reference; and U.S. Patentapplication Ser. No. 60/276,870, filed Mar. 16, 2001, incorporated byreference.

Example 1 Preparation of3-(5-carboxypentyl)-2,3-dimethyl-5-sulfoindolium, inner salt (Compound1)

A mixture of 25 g of ethyl 2-methylacetoacetate, 64 mL of a 21% sodiumethoxide solution in ethanol and 34 mL of ethyl 6-bromohexanoate isrefluxed in 200 mL of ethanol overnight. The mixture is filtered andsolvent is evaporated. The residue is partitioned between 1 M HCl andchloroform. The organic layer is dried over magnesium sulfate andpurified on silica gel using 1:10 ethyl acetate/hexanes as eluant toyield 22 g of ethyl 2-(5-carbethoxypentyl)-2-methylacetoacetate.

The acetoacetate thus obtained is dissolved in 300 mL of methanol. Asolution of 10 g NaOH in 100 mL water is added. The mixture is heated at50° C. overnight. The solution is reduced to ˜50 mL, acidified to ˜pH 1,and extracted with ethyl acetate. The organic layer is dried over MgSO₄and evaporated to yield 13.5 g of 7-methyl-8-oxononanoic acid. Thenonanoic acid is refluxed in 110 mL of acetic acid with 13.5 g of4-hydrazinobenzenesulfonic acid for 5 hours. The acetic acid isevaporated and the product is purified on silica gel to yield 23 g ofthe product.

Example 2 Preparation of2,3-dimethyl-3-(5-carboxypentyl)-5-sulfo-1-(3-sulfopropyl)indolium,sodium salt (Compound 2)

To a methanol solution of 11 g of Compound 1 is added 3.4 g of anhydroussodium acetate. The mixture is stirred for five minutes. The solvent isevaporated. The resulting sodium salt is heated with 24.4 g ofpropanesultone at 110° C. for 1 hour to generate the product.

Example 3 Preparation of5-sulfo-1-(3-sulfopropyl)-2,3,3-trimethylindolium, sodium salt (Compound3A) and 5-sulfo-1-(3-sulfopropyl-1,2,3,3-tetramethylindolium, sodiumsalt (Compound 3B)

To 15 g of 5-sulfo-2,3,3-trimethylindolium, inner salt (Mujumdar, et alBIOCONJUGATE CHEMISTRY 4, 105 (1993)) in 60 mL of methanol is added 5.67g sodium acetate. After 5 minutes at room temperature, the solution isevaporated. The foamy solid is pulverized, dissolved in 60 mLacetonitrile and stirred with 23 g propanesultone for 15 min. Followingevaporation of the solvent, the residue is dried at 110° C. to yield5-sulfo-1-(3-sulfopropyl)-2,3,3-trimethylindolium, sodium salt (Compound3A). 5-sulfo-1,2,3,3-tetramethylindolium (Compound 3B) is preparedsimilarly except that methyl p-toluenesulfonate is used instead ofpropanesultone.

Example 4 Preparation of2-(4-anilinobutadienyl)-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolium,sodium salt (Compound 4)

Compound 3A (15 g) is heated with 21.5 g malonaldehyde dianilhydrochloride and 0.4 mL triethylamine in 200 mL of acetic acid at 110°C. for one hour. The solvent is evaporated and the residue is purifiedon silica gel to yield 1.39 g of the product.

Example 5 Preparation of2-(anilinovinyl)-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolium, sodiumsalt (Compound 5)

A mixture of 5 g of Compound 3A, 2.72 g of N,N′-diphenylformamidine and0.52 mL of acetic anhydride is heated at 150° C. for 30 minutes, thenevaporated and the residue purified on silica gel.

Example 6 Preparation of2-(4-anilinobutadienyl)-5-sulfo-1,3,3-trimethylindolium, inner salt(Compound 6)

The procedure is the same as used to prepare Compound 4, except thatCompound 3B is used instead of Compound 3A.

Example 7 Preparation of3-(5-carboxypentyl)-5-sulfo-1,2,3-trimethylindolium, inner salt(Compound 7)

The compound is prepared by heating Compound 1 with 6 equivalents ofmethyl p-toluenesulfonate at 100° C. for 1.5 hours. The crude product isprecipitated with ethyl acetate.

Example 8 Preparation of Compound 8

Compound 2 (1 g) and Compound 4 (1.5 g) are combined with 0.84 mLtriethylamine and 0.5 mL acetic anhydride. The mixture is stirred atroom temperature for 1 hour, then evaporated and the residue is purifiedby HPLC.

Example 9 Preparation of Compound 9

To 55 mg of Compound 8 in 1 mL of DMF is added 0.034 mL of triethylamineand 21 mg of 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate.The mixture is stirred at room temperature for 30 minutes and evaporatedto yield the succinimidyl ester.

Example 10 Preparation of Compound 10

To Compound 9 in acetonitrile is added 3 equivalents of triethylamineand 1.2 equivalents anhydrous hydrazine. The mixture is stirred atambient temperature for 15 minutes. The product is precipitated with 4volumes of ethyl acetate and purified by HPLC.

Example 11 Preparation of Compound 11

To Compound 9 in acetonitrile at room temperature is added 4 equivalentsof triethylamine and 1.2 equivalents of N-(2-aminoethyl)maleimide,trifluoroacetic acid salt. The mixture is stirred at ambient temperaturefor 15 minutes. The product is precipitated with 4 volumes of ethylacetate and purified by HPLC.

Example 12 Preparation of Compound 12

To 6 mmole of Compound 2 is added 2 g of Compound 5 in 20 mL DMF, 4.2 mLtriethylamine, and 1.8 mL of acetic anhydride. The reaction is stirredat room temperature for one hour, then evaporated and the residue ispurified by HPLC.

Example 13 Preparation of Compound 13

The procedure is similar to that used to prepare Compound 9, usingCompound 12 in place of Compound 8.

Example 14 Preparation of Compound 14

The compound is prepared in DMF by mixing one equivalent each ofCompound 4 and Compound 7, followed by addition of four equivalents oftriethylamine and 1.5 equivalents of acetic anhydride. After stirring atroom temperature for 2 hours and evaporation, the residue is purified byHPLC.

Example 15 Preparation of Compound 15

To a mixture of 0.27 g Compound 6 and 0.6 mmoles Compound 7 in 8 mL ofDMF is added 0.42 mL triethylamine and 0.1 mL acetic anhydride. Themixture is stirred at room temperature for two hours then evaporated andthe residue is purified by HPLC.

Example 16 Preparation of Compound 16

Ethyl 2-methylacetoacetate is alkylated with 6-benzoyloxy-1-bromohexanein the presence of 1.2 equivalents of sodium hydride in THF and theresulting product was hydrolyzed and decarboxylated in aqueous NaOH asin example 1 to generate the desired 9-hydroxy-3-methyl-2-nonanone. Thenonanone is then heated at reflux with 1 equivalent of4-hydrazinebenzenesulfonic acid in acetic acid to generate3-(6-hydroxyhexyl)-2,3-dimethyl-5-sulfoindolium, inner salt. The hydroxyagain is protected as an benzoyloxy group and this intermediate is thentransformed to the protected form of target compound as in example 8.The benzoyl protecting group is then removed by dilute NaOH.

Example 17 Preparation of Compound 17

The intermediate is prepared as in Example 16, except that6-t-butoxycarbonyloxy-1-bromohexane is used instead of6-benzoyloxy-1-bromohexane. The t-BOC protecting group is removed withtrifluoroacetic acid at room temperature after formation of the targetdye.

Example 18 Preparation of Compound 18

To 1 mmole 2-methyl-1-(3-sulfopropyl)-benzothiazolium, inner salt (fromheating of one equivalent each of propanesultone and2-methylbenzothiazole at 110° C. for one hour) and 150 mg of Compound 31in 5 mL of DMF is added 0.28 mL of triethylamine and 0.1 mL aceticanhydride. The reaction is stirred at room temperature for one hour,then evaporated and the residue is purified by HPLC.

Example 19 Preparation of Compound 19

Compound 19 is prepared in the same manner as Compound 18 exceptstarting with 2-methyl-6-sulfobenzothiazole, which is prepared byreaction of sulfuric acid and 2-methylbenzothiazole at room temperature.

Example 20 Synthesis of2-(6-anilinohexatrienyl)-3,3-dimethyl-5-sulfo-1-(3-sulfopropylindolium)inner salt (Compound 20)

A mixture of 1.9 g of Compound 3A and 2.85 g ofN-(5-anilino-2,4-pentadienylidene)aniline hydrochloride in 30 mL ofacetic anhydride is heated at 120° C. for 30 minutes. At the end of theperiod, 90 mL of ethyl acetate is added and the product is filtered andused as is.

Example 21 Synthesis of Compound 21

A mixture of 1.05 g of Compound 20, 2 mmoles of Compound 2, 10 mL ofDMF, 1.7 mL of triethylamine and 0.6 mL of acetic anhydride is stirredat room temperature overnight and then at 35° C. for an additional 1.5hour. 40 mL of ethyl acetate is added and the precipitate is purified byHPLC.

Example 22 Synthesis of Compound 22

The succinimidyl ester of Compound 21 (Compound 22) is prepared asdescribed in Example 9.

Example 23 Synthesis of Compound 23

A mixture of 0.37 g of2-(4-anilinobutadienyl)-3,3-dimethyl-1-(3-sulfopropyl)indolium (preparedby the reaction of trimethylindoline and propanesultone as in Example 3followed by reaction with malonaldehyde dianil hydrochloride as inExample 4), 1.35 mmoles of3-(5-carboxypentyl)-3-methyl-1-(3-sulfopropyl)indolium (prepared by thereaction of 7-methyl-8-oxononanoic acid and phenyl hydrazine, as inExample 1), 7 mL DMF, 0.42 mL triethylamine and 0.1 mL of aceticanhydride is stirred at room temperature for one hour. Ethyl acetate (30mL) is added and the precipitate is purified on silica gel to yield 55mg of Compound 23.

Example 24 Synthesis of Corresponding Activated Esters from Free Acids

The following activated esters are prepared from the corresponding freeacids, according to the method in Example 9:

Compound 24, prepared from Compound 15

Compound 25, prepared from Compound 23

Compound 26, prepared from Compound 18

Compound 27, prepared from Compound 19

Example 25 Preparation of Compound 28

The compound is prepared by quarternization of 1,1,2trimethylbenzindoleninium 1,3-disulfonate (Bioconjugate Chem., 356-362(1996)) with propanesultone and then heated with 2 equivalents ofmalonaldehyde dianil hydrochloride in acetic acid with catalytic amountof triethylamine to yield Compound 28.

Example 26 Preparation of Compound 29

The compound is prepared by stirring one equivalent each of Compound 28and3-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-indoleniniuminner salt, sodium salt in the presence of 3 equivalents oftriethylamine and one equivalent of acetic anhydride in DMF at roomtemperature for one hour to yield Compound 29.

Compound 29 is optionally converted to its corresponding succinimidylester as described in Examples 9 and 24.

Example 27 Preparation of Compound 30

For purposes of comparison with dyes of the invention (See FIG. 4),Compound 30 is prepared according to BIOCONJUGATE CHEM. 4, 105-111(1993).

Example 28 Preparation of Compound 31

A mixture of 45 mmoles of Compound 2 and 23 g of malonaldehyde dianilhydrochloride is heated to reflux in 400 mL of acetic acid with 0.65 mLof triethylamine for 1 hour. The solvent is evaporated and the residueis purified on silica gel to yield 2.4 g of the product.

Example 29 Preparation of 2,4-dimethyloxazolo[4,5-b]pyridinium tosylate(Compound 41)

2-Amino-3-hydroxypyridine (14.48 g) is triacetylated by heating with 3equivalents of acetic anhydride at 120-130° C. for 4 hours to yield,after silica gel column purification, 10.3 g of3-acetoxy-2,2-diacetylimidopyridine. This compound is heated for 2 daysat 65° C. with 3 equivalents of methyl tosylate to yield 7 g of3-acetoxy-1-methyl-2-acetimido-1,2-dihydropyridine, p-toluenesulfonicacid salt. The 2-methyloxazolo[4,5-b]pyridine is then generated in situwhen this dihydropyridine is treated with triethylamine.

Example 30 Preparation of2-methyl-4-(3-sulfopropyl)-oxazolo[4,5-b]pyridinium, inner salt(Compound 42)

Compound 42 is prepared analogously to Compound 41 (Example 30), exceptthat propanesultone is used rather than methyl tosylate.

Example 31 Preparation of Compounds 43-46

3-Methyl-2-methylthiooxazolopyridinium tosylate is prepared by heatingthe corresponding 2-methylthiooxazolopyridines (M. Y. Chu-Moyer and R.Berger, J. Org. Chem., 60, 5721-5725 (1995)) with one equivalent ofmethyl tosylate at 100-110° C. for one hour. Derivatives preparedsimilarly include:

4-methyl-2-methylthiooxazolo[4,5-b]pyridinium tosylate

6-methyl-2-methyothiooxazolo[5,4-c]pyridinium tosylate

7-methyl-2-methylthiooxazolo[5,4-b]pyridinium tosylate

4-methyl-2-methylthiooxazolo[4,5-b]quinolinium tosylate Example 32Preparation of 2,4-dimethyl-5-methoxyoxazolo[4,5-b]quinolinium tosylate(Compound 47)

2-Amino-3-hydroxy-8-methoxyquinoline is prepared according to M. Y.Chu-Moyer (as above) starting from 2-amino-8-methoxyquinoline. Thiscompound is treated with 3 equivalents of acetic anhydride in pyridine,and the reaction mixture is heated from room temperature to 120° C. andstirred overnight. The 2-methyl-5-methoxyoxazolo[4,5-b]quinoline thusgenerated is then heated with 3 equivalents of methyl tosylate at 70° C.for 8 hours to give the desired product.

Example 33 Preparation of 2,4-dimethyloxazolo[4,5-b]quinolinium tosylate(Compound 48)

A mixture of 0.6 g of 2-amino-3-hydroxyquinoline, 3.8 mL oftrimethylorthoacetate and 0.1 g of p-toluenesulfonic acid is heated at60° C. for 7 hours. The reaction mixture is then diluted with ethylacetate, washed with sodium bicarbonate, and purified by columnchromatography on silica gel to yield 0.28 g of2-methyloxazolo[4,5-b]quinoline. The quinoline is heated with oneequivalent of methyl tosylate at 70° C. for one hour to generate theproduct.

Example 34 Preparation of 3-methyl-2-methylthiothiazolo[4,5-b]pyridiniumtosylate (Compound 49)

To 0.626 g of 2-methylthiothiazolo[4,5-b]pyridine (Smith, et al. SULFURLETTERS, 17, 197-216 (1994)) is added 0.71 g of methyl tosylate and themixture is heated at 120° C. for one hour. After cooling to roomtemperature, 10 mL of ethyl acetate is added and the mixture is stirredfor 30 minutes. The supernatant liquid is decanted and the product isrecovered as an oily layer.

Example 35 Preparation of2,3,3,7-tetramethyl-3H-pyrrolo[2,3-b]pyridinium tosylate (Compound 50)

The compound is prepared according to a literature procedure (Ficken etal., J CHEM. SOC. 3202 (1949)).

Example 36 Preparation of7-(3-sulfopropyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium, innersalt (Compound 51)

A mixture of 9 g of 2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine and 20.6 gof propanesultone is heated at 60° C. for 3 hours. The reaction mixtureis then dissolved in 100 mL of acetonitrile and 300 mL of ethyl acetateis added. The resulting sticky solid is again stirred in 300 mL of ethylacetate to yield 22 g of the product.

Example 37 Preparation of5-bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine (Compound 52)

To 40 g of 2,5-dibromopyridine in 200 mL of 2-methoxyethanol is added 53mL of anhydrous hydrazine and the mixture is heated at 110° C. for 3hours to generate the 5-bromo-2-hydrazinopyridine. A mixture of 10 g ofthis hydrazinopyridine is heated at reflux overnight with 11 mL of3-methyl-2-butanone in 40 mL of benzene equipped, using a condenserequipped with a Dean-Stark trap. All of the volatile components areremoved under reduced pressure and the resulting residue is heated in 62g of polyphosphoric acid at 140° C. for 45 minutes. The reaction mixtureis poured into water, neutralized with sodium hydroxide and extractedwith ethyl acetate. The resulting crude residue is purified bychromatography on silica gel, eluting with 1:1 ethyl acetate/hexanes, toyield 1.44 g of the product.

Example 38 Preparation of5-bromo-7-(3-sulfopropyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium,inner salt (Compound 53)

A mixture of 1 g of 5-bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridineand 1.54 g of propanesultone is heated at 65° C. for 2 hours. Ethylacetate is added and the resulting mixture is stirred at roomtemperature overnight to yield 2.26 g of the product.

Example 39 Preparation of Compounds 54 and 55

2-(4-Anilinobutadienyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,inner salt and2-(4-anilinobutadienyl)-1-(3-sulfopropyl)-3,3-dimethyl-5-sulfoindolinium,potassium salt (Compounds 54 and 55, respectively) are preparedaccording to a literature procedure (Mujumdar, et al, BIOCONJUGATECHEMISTRY 4, 105-111 (1993))

Example 40 Preparation of2-(4-anilinobutadienyl)-3-carboxypentyl-benzothiazolium bromide(Compound 58)

The desired compound is prepared by heating 3.44 g of3-carboxypentyl-2-methylbenzothiazolium bromide, 5.14 g of malonaldehydedianil hydrochloride and 2 mL of acetic anhydride in 30 mL of aceticacid for 6 hours.

Example 41 Preparation of Succinimidyl Esters of Carboxylic Acids

Succininimidyl ester derivatives are typically prepared from thecorresponding carboxylic acids using the2-succinimido-1,1,3,3-tetramethyl uronium tetrafluoroborate (Bannwarthet al. TETRAHEDRON LETT. 1157-1160 (1991)) and either triethylamine ordiisopropylethylamine. Succinimidyl ester derivatives are also readilyprepared by coupling a carboxylic acid derivative toN-hydroxysuccinimide using an activating agent such as a carbodiimide.

Example 42 Preparation of7-(carboxypentyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium bromide(Compound 59)

A mixture of 0.54 g of 2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine and1.32 g of 6-bromohexanoic acid is heated at 120° C. for one hour. Ethylacetate (10 mL) is added, and the reaction mixture is heated at refluxfor 15 minutes, then cooled to room temperature. The supernatant liquidis decanted to yield the product.

Example 43 Preparation of Compound 60

To one equivalent each of2-methyl-4-(3-sulfopropyl)-oxazolo[4,5-b]pyridinium, inner salt(Compound 42) and2-(4-anilinobutadienyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,inner salt (Compound 54) in DMF are added 4 equivalents of triethylamineand 2 equivalents of acetic anhydride. After stirring at roomtemperature for 2 hours, ethyl acetate is added to precipitate the crudeproduct, which is purified by chromatography on silica gel.

Example 44 Preparation of Compound 61

To 0.32 g of 3-acetoxy-2-acetimido-1-(sulfopropyl)-1,2-dihydropyridine,inner salt and 0.4 g of2-(anilinovinyl)-3-(carboxypentyl)benzothiazolium tosylate in 5 mL ofmethylene chloride are added 0.14 g of triethylamine and 0.1 mL ofacetic anhydride. The reaction mixture is stirred at room temperaturefor two hours. Ethyl acetate (8 mL) is added and the supernatantsolution is decanted. The sticky residue is recrystallized from amixture of DMF and ethyl acetate to yield the desired product.

Example 45 Preparation of Compound 62

To a 0.05 mmole solution of2,4-dimethyl-5-methoxyoxazolo[4,5-b]quinolinium tosylate in 2.5 mL ofacetonitrile is added 20 mg of2-(anilinovinyl)-3,3-dimethyl-1-(carboxypentyl)-5-sulfoindoleniumiodide, 0.014 mL of triethylamine and 5 μL of acetic anhydride. Thereaction mixture is heated at 60° C. overnight. The product is purifiedon a silica gel column.

Example 46 Preparation of Compound 63

To 0.28 g of 2,3,3,7-tetramethyl-H-pyrrolo[2,3-b]pyridinium iodide and0.3 g of2-(4-anilinobutadienyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,inner salt in 3 mL of DMF at room temperature are added 0.2 mL of aceticanhydride and 0.42 mL of triethylamine. The reaction mixture is stirredat room temperature for 2 hours then the crude product is purified on asilica gel column.

Example 47 Preparation of Compound 64

To an equivalent each of3-acetoxy-1-methyl-2-acetimido-1,2-dihydropyridine, p-toluenesulfonicacid salt and2-(4-anilinobutadienyl)-3-(5-carboxypentyl)-6-sulfobenzothiazolium,inner salt in DMF are added 3 equivalents of triethylamine and oneequivalent of acetic anhydride to generate the desired product, which ischromatographically purified.

Example 48 Preparation of Compound 65

To a mixture of 0.35 g of7-(carboxypentyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium bromideand 92 mg of2-(4-anilinobutadienyl)-1-(3-sulfopropyl)-3,3-dimethyl-5-sulfoindolinium,potassium salt in 5 mL of DMF is added 0.28 mL of triethylamine and 0.06mL of acetic anhydride. The mixture is stirred at room temperature for1.5 hour. Ethyl acetate (20 mL) is added and the crude precipitate ispurified on a silica gel column.

Example 49 Preparation of Compound 66 and Compound 67

A mixture of 0.32 g of2-(4-anilinobutadienyl)-3-carboxypentyl-3-methyl-5-sulfo-1-sulfopropylindolinium,sodium salt, 0.75 g of5-bromo-7-(3-sulfopropyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium,inner salt, 0.36 mL of triethylamine and 0.1 mL of acetic anhydride isstirred in 13 mL of DMF at room temperature for 1 h. 50 mL of ethylacetate is added then the crude solid is filtered and purified by HPLC.A succinimidyl ester derivative (Compound 67) is prepared according tothe methods provided in Example 41.

Example 50 Preparation of Compound 68 and Compound 69

The compound is prepared in a similar manner as Compound 66 except that7-(3-sulfopropyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium, innersalt is used. The crude product is purified by HPLC. A succinimidylester derivative (Compound 69) is prepared according to the methodsprovided in Example 41.

Example 51 Preparation of Compound 70 and Compound 71

A mixture of 100 mg of2-(anilinovinyl)-3,3-dimethyl-5-sulfo-1-sulfopropylindolenium, innersalt, potassium salt, 2 mmole of7-(5-carboxypentyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium bromide,5 mL of DMF, 0.2 mL of triethylamine and 0.12 mL of acetic anhydride isstirred at room temperature for 2 hours. At the end of the period, 20 mLof ethyl acetate is added and the crude precipitate is further purifiedon a silica gel column. The succinimidyl ester of this dye (Compound 71)is prepared as in Example 41.

Example 52 Preparation of Compound 72

A mixture of 150 mg of2-(anilinobutadienyl)-3-(3-carboxypropyl)-5-sulfo-1-sulfopropylindolenium,inner salt, potassium salt, 160 mg of2,3,3,7-tetramethyl-3H-pyrrolo[2,3-b]pyridinium tosylate, 2 mL of DMF,0.13 mL of triethylamine and 0.08 mL of acetic anhydride is heated at40° C. for 30 minutes. Volatile components are evaporated and the cruderesidue is purified on a silica gel column.

Example 53 Preparation of Compound 73 and Compound 74

A solution of 3.2 g of7-(3-sulfopropyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium, innersalt and 4 g of2-(4-anilinobutadienyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,inner salt in 40 mL of DMF, 3.4 mL of triethylamine and 2 mL of aceticanhydride is stirred at room temperature for 4 hours. At the end of theperiod, 700 mL of acetonitrile is added and the crude solid is recoveredby filtration and purified by HPLC. The succinimidyl ester derivative(Compound 74) is generated by the methods provided in Example 41.

Example 54 Preparation of Compound 75

To a mixture of 30 mg of the succinimidyl ester Compound 74 (Example 53)and 10 mg of N-(2-aminoethyl)maleimde, trifluoroacetic acid salt in 2 mLof acetonitrile is added 0.015 mL of triethylamine is added and themixture stirred at room temperature for 30 minutes. Then 6 mL of ethylacetate is added and the solid filtered to yield the product.

Example 55 Preparation of Compound 76 and Compound 77

To 5 g of5-bromo-7-(3-sulfopropyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium,inner salt and 2.5 g of2-(4-anilinobutadienyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,inner salt in 20 mL of DMF at room temperature are added 5.8 mL oftriethylamine and 3.5 mL of acetic anhydride. The reaction mixture isstirred at room temperature for 5 hours. At the end of the period, 140mL of ethyl acetate is added, and the crude solid is collected byfiltration and then purified by HPLC. The succinimidyl ester derivative(Compound 77) is generated by the methods provided in Example 41.

Example 56 Preparation of Compound 78

A mixture of 60 mg of the succinimidyl ester Compound 77 (Example 55)and 18 mg of N-(2-aminoethyl)maleimide, trifluoroacetic acid in 2 mL ofacetonitrile at room temperature and 0.027 mL of triethylamine isstirred at ambient temperature for 30 minutes. Then 6 mL of ethylacetate is added and the product is collected by filtration.

Example 57 Preparation of Compound 79

To 1.7 g of 2,3-dimethyl-6-sulfobenzothiazolium tosylate in 20 mL ofpyridine at room temperature is added 1.03 mL of methyl5-chloro-5-oxovalerate. The mixture is heated at 50-60° C. for 3 hours.The pyridine solvent is removed under reduced pressure, and the reactionis worked up with chloroform and brine, and purified by silica gelcolumn to yield 0.92 g of2-(5-methoxycarbonyl-2-oxopentylidene)-3-methyl-3H-benzothiazole. Amixture of 0.45 g of this benzothiazole and 0.45 g of phosphorousoxychloride in 5 mL of dichloroethane is heated at reflux for 2 hours togenerate2-(2-chloro-2-methoxycarbonylpropylvinyl)-3-methylbenzothiazoliumchloride. The volatile components are evaporated and the crude chlorideis used without further purification. The crude chloride and 0.45 g of2,3,3,7-tetramethyl-3H-pyrrolo[2,3-b]pyridinium tosylate is stirred in 5mL of dichloroethane in the presence of 0.45 mL of triethylamine for 2hours. The volatile components are removed under reduced pressure, andthe residue is dissolved in 5 mL of methanol and added dropwise to asolution of 4.5 g of sodium iodide in 30 mL water. The sticky solid ispurified on a silica gel column to yield Compound 79.

Example 58 Preparation of Compound 80

To 30 mg of the succinimidyl ester Compound 74 (Example 53) in 3 mL ofacetonitrile, 0.01 mL of triethylamine and 107 mg of anhydrous hydrazineare added. The reaction mixture is stirred for 15 minutes then 12 mL ofethyl acetate is added to precipitate the product.

Example 59 Preparation of Compound 81

The intermediate2-(6-anilinohexatrienyl)-1-5-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,inner salt is prepared according to literature procedure (Licha et al.TET. LETT., 1711-1715 (2000)). To 0.1 g of7-(3-sulfopropyl)-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridinium, innersalt and 1 equivalent of2-(6-anilinohexatrienyl)-1-5-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,inner salt in 3 mL of DMF, 0.2 mL of triethylamine and 2 equivalents ofacetic anhydride are introduced. The mixture is stirred at roomtemperature overnight and 30 mL of ethyl acetate is then added toprecipitate the crude product. Purification by column chromatography onsilica gel produces the pure product (Ex/Em (MeOH) 750 nm/800 nm,quantum yield=0.12).

Example 60 Preparation of Compound 82 and Compound 86

6-Hydrazinonaphthalene 1,3-disulfonate (Bioconj. Chem., 356-362 (1996))is heated with 7-methyl-8-oxononanoic acid in acetic acid to generatethe 1-carboxypentyl-1,2-dimethyl-6,8-disulfobenzindoline. Thebenzindoline is quarternized with propane sultone and chain elongatedwith malonaldehyde dianil hydrochloride to yield the2-(4-anilinobutadienyl) derivative (Compound 82) which is then reactedwith compound 53 in the presence of acetic anhydride and triethylamineto yield the desired product.

Example 61 Preparation of Compound 83

A mixture of 100 mg of 4-acetylcyclohexanecarboxylic acid and 120 mg ofhydrazinobenzenesulfonic acid is refluxed in 5 mL of acetic acid toobtain the desired compound.

Example 62 Preparation of Compound 84

A mixture of 2-(4-carboxyphenyl)-butan-2-one andhydrazinobenzenesulfonic acid in acetic acid is refluxed for 3 hours toobtain the desired product.

Example 63 Preparation of Compound 85

A mixture of 0.85 g of 2-hydrazinopyridine and 1.6 g of7-methyl-8-oxononanoic acid is refluxed in 10 mL of benzene overnight.The volatile components are evaporated and the residue is heated with0.2 g of zinc chloride at 250° C. for one hour to obtain the desiredproduct.

Example 64 Preparation of a Phalloidin Dye-Conjugate

To aminophalloidin p-toluenesulfonate (3.5 mg, 4 μmol) and thesuccinimidyl ester derivative Compound 9 or 69 (6.0 mg, 5 μmol) in DMFis added N,N-diisopropylethylamine (2 μL, 11 μmol). The mixture isstirred at room temperature for 3 hours. To this solution is added 7 mLof diethyl ether. The solid is collected by centrifugation. The crudeproduct is purified on SEPHADEX LH-20, eluting with water, followed bypreparative HPLC to give the pure phalloidin conjugate. The product isan effective stain for F-actin filaments in fixed-cell preparations.

Example 65 Preparation of a Drug Dye-Conjugate

A fluorescent dopamine D₂ antagonist is prepared as follows: To 10 mg ofN-(p-aminophenethyl)spiperone (Amlaiky et al., FEBS LETT 176, 436(1984)), and 10 μL N,N-diisopropylethylamine in 1 mL of DMF is added 15mg of Compound 24 or 69. After 3 hours, the reaction mixture is pouredinto 5 mL ether. The precipitate is centrifuged, then purified bychromatography on silica gel using 10-30% methanol in chloroform.

Example 66 Preparation of Protein Dye-Conjugates

A series of dye conjugates of goat anti-mouse IgG (GAM), goatanti-rabbit IgG (GAR), streptavidin, transferrin and other proteins,including Conconavalin A, R-phycoerythrin (R-PE) and allophycocyanin(APC) are prepared by standard means (Haugland et al., METH. MOL. BIOL.45, 205 (1995); Haugland, METH. MOL. BIOL. 45, 223 (1995); Haugland,METH. MOL. BIOL. 45, 235 (1995); Haugland, CURRENT PROTOCOLS IN CELLBIOLOGY 16.5.1-16.5.22 (2000)) using Compound 9 or 74 and amono-succinimidyl ester derivative of the Cy5 dye (Amersham-PharmaciaBiotech).

The typical method for protein conjugation with succinimidyl esters ofthe invention is as follows. Variations in ratios of dye to protein,protein concentration, time, temperature, buffer composition and othervariables that are well known in the art are possible that still yielduseful conjugates. A solution of the protein is prepared at ˜10 mg/mL in0.1 M sodium bicarbonate. The labeling reagents are dissolved in asuitable solvent such as DMF at ˜10 mg/mL. Water is a suitable solventfor many dyes of the invention. Predetermined amounts of the labelingreagents are added to the protein solutions with stirring. A molar ratioof 10 equivalents of dye to 1 equivalent of protein is typical, thoughthe optimal amount varies with the particular labeling reagent, theprotein being labeled and the protein's concentration, and is determinedempirically.

When optimizing the fluorescence yield and determining the effect ofdegree of substitution (DOS) on this brightness, it is typical to varythe ratio of reactive dye to protein over a several-fold range. Thereaction mixture is incubated at room temperature for one hour or on icefor several hours. The dye-protein conjugate is typically separated fromfree unreacted reagent by size-exclusion chromatography, such as onBIO-RAD P-30 resin equilibrated with phosphate-buffered saline (PBS).The initial, protein-containing colored band is collected and the degreeof substitution is determined from the absorbance at the absorbancemaximum of each fluorophore, using the extinction coefficient of thefree fluorophore. The dye-protein conjugate thus obtained can besubfractionated to yield conjugates with higher, lower or more uniformDOS.

A solution of the desired protein is prepared at 10 mg/mL in 0.1 Msodium bicarbonate. The labeling reagents are dissolved in DMF at 10mg/mL. Predetermined amounts of the labeling reagents are added to theprotein solutions with stirring. A molar ratio of 10 equivalents of dyeto 1 equivalent of protein is typical, though the optimal amount varieswith the particular labeling reagent, the protein being labeled and theprotein's concentration, and is determined empirically. The reactionmixture is incubated at room temperature for one hour, or on ice forseveral hours. The dye-protein conjugate is typically separated fromfree unreacted reagent by size-exclusion chromatography on BIO-RAD P-30resin equilibrated with PBS. The initial, protein-containing coloredband is collected and the degree of substitution is determined from theabsorbance at the absorbance maximum of each fluorophore, using theextinction coefficient of the free fluorophore.

TABLE 5 Fluorescence of Protein Conjugates of Compound 69 Protein DOS*Quantum Yield† Goat anti-Mouse IgG 3.7 0.47 Streptavidin 4.5 0.85 WheatGerm Agglutinin 3.1 0.32 Goat anti-Rabbit IgG 4.4 0.5 (highlycross-absorbed) Goat anti-Chicken IgG 4.5 0.33 Rabbit anti-Mouse IgG 3.00.67 Goat anti-Mouse IgG 4.4 0.63 (highly cross-absorbed Goatanti-Guinea Pig IgG 5.1 0.33 Protein A (MR = 4)‡ 2.1 0.64 Protein A (MR= 8)‡ 4.9 0.39 *Extinction coefficients are determined for the freecarboxylic acid in aqueous solution †Quantum yield measured relative toDDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridine-2-one) ‡MR is theapproximate molar ratio of the dye to protein following conjugation

TABLE 6 Fluorescence of Protein Conjugates of Compound 67 Protein DOS*Quantum Yield Goat anti-Mouse IgG 5.9 0.15 Streptavidin 4.8 0.66 Goatanti-Rabbit IgG 4.9 0.26 (highly cross-absorbed) Goat anti-Chicken IgG —— Rabbit anti-Mouse IgG 6.0 0.31 *Extinction coefficients are determinedfor the free carboxylic acid in aqueous solution

Protein conjugates of antibody fragments, of other avidins and of otherproteins are prepared and analyzed similarly.

Example 67 Fluorescent Labeling of Periodate-Oxidized Proteins

Two samples of 5 mg each of goat IgG antibody in 1 mL of 0.1 M acetate,0.135 M NaCl, pH 5.5, are treated with 2.1 mg of sodium metaperiodate onice, for 1 and 2 hours, respectively. The reactions are stopped byaddition of 30 μL ethylene glycol. The antibodies are purified on aMATREX GH 25 column (1 cm×30 cm) packed in PBS pH 7.2. One-tenth volumeof 1 M sodium bicarbonate is added to raise the pH and Compound 17 or 80is added at a molar ratio of dye to protein of 50:1. The reaction isstirred for 2 hours at room temperature. Sodium cyanoborohydride isadded to a final concentration of 10 mM and the reaction is stirred for4 hours at room temperature. The antibody conjugates are purified bydialysis and on CELLUFINE MATREX GH 25 columns as described above.Antibodies that are oxidized for 1 hour typically yield a degree ofsubstitution of 1 mole of dye per mole of IgG. Antibodies that areoxidized for 2 hours typically yield a DOS of approximately 2 mole ofdye per mole of IgG. Periodate-oxidized proteins in gels and on blotscan also be labeled, essentially as described in Estep T. N. and MillerT J., (Anal Biochem 157, 100-105 (1986))

Example 68 Total Fluorescence of Selected Dye-Protein Conjugates as aFunction of Degree of Substitution (DOS)

The conjugates of Compound 9 exhibit equal or greater fluorescence thanthe conjugates of Cy5 dye at similar DOS when conjugated to a widevariety of proteins (Table 3). Protein conjugates are prepared (asdescribed in Example 66) at varying DOS and compared for brightness(total-fluorescence, TF) and relative quantum yield (RQY; definition atbottom of Table 3). Total fluorescence is proportional to the overallbrightness of the bioconjugate, and is defined as the product of the RQYand DOS: TF=RQY×DOS.

Example 69 Total Fluorescence of Selected Dye-Protein Conjugates as aFunction of Degree of Substitution

Table 3 confirms the report of Gruber et al. (BIOCONJUGATE CHEM. 11, 696(2000)) of heavy quenching of the fluorescence of Cy5 conjugates (ateven moderate DOS). For instance, comparing GAM IgG Compound 9 at ˜DOS4.2 with a GAM IgG Cy5 at DOS˜4.8 (see Table 3), reveal that theCompound 9 bioconjugate is approximately 5.0/0.4 brighter (12.5×) thanthe Cy5 bioconjugate. This type of pattern is observed for all of theproteins in Table 3. In general, the higher the DOS, the brighterCompound 9 bioconjugates are relative to the Cy5 bioconjugates,although, as can be seen, the Compound 9 bioconjugates are brighter atall DOS tested.

The decrease in the RQY of the Cy5 bioconjugates is found to beaccompanied by an increase in the 600-nm absorbance band relative to the650-nm absorbance band. This effect is true for all of the bioconjugateslisted in Table 3. FIG. 2 shows a direct comparison of the absorptionand fluorescence emission of the Compound 9 and Cy5 conjugate of GAM IgGat nearly equivalent DOS (Compound 9-GAM DOS˜4.2, Cy5-GAM DOS˜4.8). The600 nm absorbance band is always much lower in extinction for Compound 9than for an equivalently labeled Cy5 derivative. One can clearly see theincrease in 600 nm absorbance for the Cy5 bioconjugate relative to theCompound 9 bioconjugate (FIG. 2) for all of the bioconjugates tested(Table 3). The increase in extinction of the 600 nm band is alwaysassociated with a large quenching of the fluorescence. This result iscompletely supportive of the work of Gruber et. al. (BIOCONJUGATE CHEM.11, 696 (2000) who observed a similar correlation of an increasedabsorbance at 600 nm and a large decrease in fluorescence intensity.This general observation has now been confirmed with several otherproteins (Table 3).

The Cy5 and Compound 9 derivatives of GAM (from FIG. 2) are examinedusing both absorbance spectroscopy and fluorescence excitationspectroscopy (FIG. 3). The fluorescence excitation spectra (emissionwavelength=725 nm) of each derivative is normalized to its absorbancespectra at 660 nm. Dramatically clear in this figure is how the Cy5 GAM600 nm absorbance band does not emit fluorescence, as evidenced by thelarge difference between the excitation and absorbance in this region ofthe spectrum. These data support the work of Gruber et al. (BIOCONJUGATECHEM. 11, 696 (2000)), who saw similar changes in absorbance for Cy5derivatized antibodies. The absorbance and excitation spectra ofCompound 9 nearly overlap in this region. When this same Cy5-GAMantibody is dissolved in 6.0 M guanidinium hydrochloride (pH=7.5), the600 nm absorbance band greatly decreases, the 650 nm absorbance bandincreases, and the overall fluorescence intensity increasesdramatically. This result indicates that it is the behavior of the Cy5derivative on the bioconjugate native structure, that causes the largedecrease in fluorescence. A similar result was obtained for Cy5derivatives of nuclease-digested DNA (Example 94, FIG. 13). Compound 9bioconjugates have absorbance and excitation spectra that are much moreclosely alligned (e.g., FIG. 3), and hence show a drastically reducedamount of fluorescence quenching.

Example 70 Comparison of Two Structural “Cy5-Like” Isomers (Compound 30and Compound 24) which Reveal the Origin of the Shift in Extinction fromthe 650 nm to 600 nm Bands on Bioconjugates of Cy5

In order to better understand the origin of the anomalous absorptioneffects of Cy5 bioconjugates and the much smaller effect with Compound9, two isomers of Cy5 were synthesized and conjugated to GAR at variousDOS (Compound 30 and Compound 24). FIG. 4 is a direct comparison ofabsorbance properties of these two isomers at DOS's of approximately2.8, 4.3, and 5.5 on GAR. The only difference between the chemicalstructures of Compounds 30 and 24 is the change in position of thereactive moiety from position 1 (Cy5 position) to position 3 (shared byall dyes of the invention) of the indolium ring. One can see that thischemical change has resulted in a drastic improvement of the behavior ofthe absorbance of Compound 24 over Compound 30. Compound 24 has abrighter fluorescence emission than Compound 30 at all of these testedDOS's (Table 3).

Example 71 Comparison of the Total Fluorescence of GAM Conjugates ofCompound 9 with Commercially Available Conjugates of the Cy5 Dye

Conjugates of the Cy5 dye with GAM were purchased from several sources(Table 3). Absorption spectra of each of these conjugates confirm thegeneral observations made in FIGS. 2 and 3, in that the Cy5 GAMconjugates all had much larger absorbances at 600 nm (relative to the650 nm band) than the corresponding Compound 9-GAM conjugates. Forfluorescence brightness comparisons, the proteins are adjusted toapproximately the same concentration as measured by the absorption at280 nm corrected for the contribution of dye absorption at 280 nm. Forsome commercial Cy5 bioconjugates, the protein concentration supplied bythe vendor was utilized due to the presence of 280 nm-absorbingstabilizers. The conjugates are excited at 633 nm and the fluorescenceemission spectrum measured. The results in Table 3 confirm the superiorfluorescence brightness (TF) of GAM conjugates of Compound 9 comparedwith the commercially available Cy5 conjugates of GAM.

Example 72 Comparison of the Total Fluorescence of GAM Conjugates ofCompound 9 with Commercially Available Conjugates of the Cy5 Dye withGAM Using Flow Cytometry

The fluorescence intensity comparisons from Examples 68 and 69 confirmspectroscopically the greater fluorescence emission of bioconjugates ofCompound 9 compared with the comparable Cy5 bioconjugates. Thefluorescence intensity comparisons described in this example, revealthat the increased brightness is also apparent in experiments performedon a flow cytometer. The flow cytometer intensity comparisons do notrely on the same type of RQY, DOS, and TF calculations required for thespectroscopically based fluorescence intensity comparisons.

Human peripheral blood is drawn in a sodium heparin tube. One hundred μLof blood is added to a Falcon tube. The blood is blocked with 5 μL ofmouse monoclonal antibodies to both human CD16 and CD32 (Caltag) for 15minutes at room temperature. The cells are washed with PBS andresuspended to 100 μL. The blood is then incubated with mouse monoclonalantibody to mouse anti-CD3 (Caltag) at the recommended concentration of0.50 μg for 30 minutes at 37 degrees Celsius. After incubation with theprimary antibody, the cells are washed and resuspended. The blood isthen incubated with GAM conjugates of Compound 9 (prepared as in Example66) and the commercial GAM conjugates of Cy5 from Jackson Laboratories(DOS˜1.9) and Amersham-Pharmacia (DOS˜11) at a concentration of 0.50 μgfor 30 minutes at 37 degrees Celsius. The red blood cells are lysed witha cell lysis buffer and centrifuged to remove the lysed red blood cells.The cell pellet is washed once with PBS and resuspended to a finalvolume of 500 μL. The samples are analyzed on a FACScan flow cytometer(BD Biosciences) exciting with a 488 nm argon-ion laser and a long-pass(>650 nm) filter. A direct comparison of GAM conjugates of Compound 9with the commercially available labeled GAM conjugates of Cy5 is shownin FIG. 5. The geometric mean of the background subtracted fluorescenceintensity for the Compound 9 conjugates of GAM is 164, whereas theCy5-GAM conjugates prepared by Jackson Laboratories andAmersham-Pharmacia are 71, and 30, respectively.

Flow cytometry studies are performed as a function of DOS for thisantibody, and it is found that at all DOS's, the conjugate of Compound 9with GAM is from 1.4× to 5.9× brighter than the commercially availableCy5 conjugates of GAM (see Table 7). The flow cytometric results supportthe conclusion from the spectroscopic characterizations (Examples 68 and69) that Compound 9 bioconjugates are brighter than the correspondingCy5 bioconjugates.

TABLE 7 (Compound 9 GAM)/ (Compound 9 GAM)/ DOS (Amersham Cy5 GAM)†(Jackson Cy5 GAM)‡ 1.83 4.84 1.92 2.36 5.7 2.26 3.94 5.8 2.26 4.36 5.862.33 7.25 3.6 1.4 †, ‡The geometric mean of the background subtractedfluorescence intensities obtained from Compound 9-labeled GAM divided bythe intensity of Amersham-Pharmacia Biotech (†) or the JacksonLaboratories (‡) Cy5 version of this same antibody.

Example 73 Comparison of the Fluorescence of Goat Anti-Rabbit IgG (GAR)Conjugates of Compound 13 and those of the Spectrally Similar CY3 Dye

GAR is labeled with Compound 13 and the CY3 reactive dyes at a varietyof degrees of substitution ranging from 1.0-12. In all cases, the GARconjugates of Compound 13 are superior in brightness to the Cy3dye-labeled GAR (at equivalent DOS). A typical example is shown in FIG.6 (DOS˜6.3). Excitation wavelength=532 nm.

Example 74 The Photostability of Compound 9 is Greater than that of Cy5Reactive Dye

Photobleaching experiments are performed in small capillary tubes at 0.5μM concentrations of Compound 9 and commercially available Cy5 reactivesuccinimidyl esters, in PBS, pH 7.5. The 40× objective of a NikonEclipse E-400 and Cy3/Cy5 filter XF92 (Omega) with a 100 W Mercury lampexcitation, is utilized. Integrated intensities are collected underconstant illumination as a function of time (FIG. 7). After 100 minutesof illumination, Compound 9 remains about 2× brighter than the Cy5 dye.

Example 75 Labeling β-Galactosidase with a Thiol-Reactive Dye

A solution of β-galactosidase, a protein rich in free thiol groups, isprepared in PBS (2.0 mg in 400 μL). The protein solution is then treatedwith a 20 mg/mL solution of the maleimide derivative Compound 11 in DMF.Unreacted dye is removed on a spin column. The degree of substitution bythe dye is estimated using the extinction coefficient of the free dye.The protein concentration is estimated from the absorbance at 280 nm,corrected for the absorbance of Compound 11 at that wavelength.

Example 76 Fluorescence Energy Transfer in Conjugates of R-phycoerythrinand Allophycocyanin

An R-phycoerythrin (R-PE) conjugate of Compound 9 or 74 is prepared asin Example 66 with a DOS sufficiently high to quench the donorfluorescence almost completely (DOS˜4-8). The resulting phycobiliproteinconjugate is excited at 488 nm and the fluorescence emission is comparedto that of unmodified R-phycoerythrin excited at the same wavelength.Highly efficient energy transfer (>99%) occurs from the protein to thefluorescent dye (FIG. 8).

Compound 22 conjugated to R-PE with a DOS of 4.7, 8.2, and 13, generatesenergy-transfer efficiencies of ˜90%, ˜99%, and ˜99.3%, respectively. Aconjugate of these complexes with streptavidin is prepared essentiallyas described by Haugland (METH. MOL. BIOL. 45, 205 (1995), supra). Thisstreptavidin conjugate retains the energy transfer properties and isuseful for cell staining in flow cytometers that utilize the argon-ionlaser for excitation.

Tandem conjugates of allophycocyanin can also be made, with longerwavelength dyes of the invention such as Compound 22. These conjugatesyield emission well beyond 700 nm when excited near 633 nm (FIG. 9).

Example 77 Staining Cells with Tandem Dye-Labeled Streptavidin

Human peripheral blood is drawn in a sodium heparin tube and treatedexactly as described in Example 72, except in this case, a biotinylatedanti-CD3 antibody (Caltag) is utilized as the primary step, andtandem-dye conjugates of Compound 9-derivatized streptavidin-R-PE andthe commercial Cy5 version of this product (Gibco Red 670) are utilizedfor detection in parallel experiments. The samples are analyzed on aFACScan flow cytometer (BD Biosciences) exciting with a 488 nm argon-ionlaser and a long-pass (>650 nm) filter. A direct comparison of thetandem streptavidin-R-PE conjugate of Compound 9 with the commerciallyavailable tandem dye-labeled streptavidin-R-PE reveals that thesignal-to-noise ratio of the tandem conjugate prepared from Compound 9is ˜4.5× brighter than the corresponding Cy5 tandem conjugate (FIG. 10).

Example 78 Labeling and Use of a Wheat Germ Agglutinin Dye-Conjugate

Wheat germ agglutinin (100 mg, EY Laboratories) is dissolved in 5 mLNaHCO₃, pH 8.3, containing 9 mg N-acetylglucosamine. To the solution isadded 9 mg of Compound 9. After 1 hour the solution is purified by gelfiltration. A degree of substitution of 2-3 dyes per molecule isdetermined from the absorption at 650 nm.

A 1 mg/mL stock solution of the resulting wheat germ agglutinin (WGA)conjugate is prepared in 0.1 M sodium bicarbonate pH 8. Staphylococcusaureus are cultured for 17 hours at 30° C. in TSB broth. Equal volumesof the TSB culture and a bovine serum albumin (BSA) solution (0.25%BSA+0.85% NaCl sterile filtered through 0.2 μm filter) are incubated atroom temperature for 15 minutes. The BSA-bacterial suspension (200 μL)is centrifuged for 2 minutes at 350×g, capturing the bacteria on afilter membrane. The cells are resuspended in 90 μL of BSA solution and10 μL of stain is added for 15 minutes. Following centrifugation, thebacteria are resuspended in BSA solution, and an aliquot is trappedbetween a slide and a glass coverslip.

The bacteria are observed on a Nikon Diaphot epifluorescence microscopeusing an appropriate band pass filter set. Images are acquired using theStar-1 cooled CCD camera and the software package supplied with thecamera is used for data analysis. Two images are collected for eachstain, each image having a 2 second exposure time. When used accordingto Sizemore et al. (U.S. Pat. No. 5,137,810) the conjugate candistinguish between Gram-positive and Gram-negative bacteria.

Example 79 Simultaneous Labeling of Actin and Tubulin in CulturedMammalian Cells

Bovine pulmonary artery cells (BPAEC) are grown to 30-50% of confluenceon glass. The cells are fixed with 3.7% formaldehyde, permeabilized with0.2% Triton X-100, and blocked with 6% BSA. The cells are incubated withmouse monoclonal anti-α-tubulin for 60 min.

A cell sample is labeled with a monoclonal mouse anti-tubulin (MolecularProbes, Inc.) and a GAM conjugate of Compound 13 for 30 min, washed, andthen incubated with the phalloidin dye-conjugate of Example 64 for anadditional 30 min. The cells are rinsed with blocking buffer and mountedin PBS pH 7.4. The stained cells display microtubules decorated withgreen fluorescence and actin filaments decorated with red fluorescence.Additionally, nuclei can be distinguished by their blue fluorescencewhen stained with DAPI.

Example 80 Preparation and Use of a Fluorescent α-BungarotoxinDye-Conjugate

α-Bungarotoxin (1 mg) in 25 μL 0.1 M NaHCO₃ is treated with 1.5equivalents of the succinimidyl ester of Compound 18 or 1.5 equivalentsof Compound 69 at room temperature for 2 hours. The product is purifiedby size exclusion, by ion exchange chromatography, and finally byreverse-phase HPLC. Staining of acetylcholine receptors and detection oftheir resulting fluorescence, although detected at a longer wavelength,is comparable to that obtained with TEXAS RED dye-conjugatedα-bungarotoxin.

Example 81 Preparation of a Fluorescent Tyramide

A 2-fold molar excess of tyramine hydrochloride is added to Compound 9in aqueous solution at room temperature followed by an excess oftriethylamine. After 30 minutes the red solid is precipitated withacetone, washed with ether and purified by preparative HPLC.

Example 82 Peroxidase-Catalyzed Deposition of a Fluorescent Tyramide

Bovine pulmonary artery cells (BPAEC) are grown to 30-50% of confluenceon glass. The cells are fixed with 3.7% formaldehyde, permeabilized with0.2% Triton X-100, and blocked with 1 mg/mL streptavidin and 1 mMbiotin. After washing, cells are exposed to ˜0.05 μg/mL of biotinylatedanti-cytochrome c oxidase (anti-COX) (Molecular Probes) then incubatedwith Streptavidin-HRP conjugate (Molecular Probes) for 60 minutes atroom temperature or 37° C. Cells are rinsed again. The sample is thenincubated with Compound 9 tyramide (synthesized as described in Example81) and examined using fluorescence microscopy (Cy5 filter set, OmegaXF47). A bright fluorescence emission localized on the mitochondria isobtained, indicating the successful molecular targeting of COX by theCompound 9 tyramide.

Example 83 Preparation of Aminodextran Dye-Conjugates

70,000 MW aminodextran (50 mg) derivatized with an average of 13 aminogroups is dissolved at 10 mg/mL in 0.1 M NaHCO₃. Compound 13 or 69 isadded so as to give a dye/dextran ratio of ˜12. After 6 hours theconjugate is purified on SEPHADEX G-50, eluting with water. Typically4-6 moles of dye are conjugated to 70,000 MW dextran.

Example 84 Preparation of Fluorescent-Dye Labeled Microspheres

Uniform microspheres are conjugated to the dyes of the invention by oneof four methods. In Method A, 1.0 μm amine-derivatized polystyrenemicrospheres are suspended at ˜2% solids in 100 mM NaHCO₃, pH 8.3 andtreated with 2 mg/mL of an amine-reactive dye. After 1 hour themicrospheres are centrifuged and washed with buffer.

In Method B, carboxylate-modified microspheres are suspended in asolution of a protein that has been conjugated to a dye of theinvention. The protein is passively adsorbed on the microspheres, andexcess protein is removed by centrifugation and washing. Microparticlesof a size that cannot be centrifuged are separated from excess proteinby dialysis through a semi-permeable membrane with a high MW cutoff orby gel filtration chromatography.

In Method C a dye-labeled protein is covalently coupled through itsamine residues to the carboxylate groups of the polymer using ethyl3-(dimethylaminopropyl)carbodiimide (EDAC).

In Method D, biotinylated microspheres are treated with a streptavidin,avidin or anti-biotin conjugate of a dye of the invention, and theconjugates are isolated as in Method B.

The larger particles can be analyzed for uniformity of staining andbrightness using flow cytometry. The microspheres can be further coupledto proteins, oligonucleotides, haptens and other biomolecules for assaysusing methods well known in the art.

Example 85 Preparation of Fluorescent Liposomes Using Dyes of theInvention

Selected dyes of the invention are sufficiently water soluble to beincorporated into the interior of liposomes by methods well known in theart (J. BIOL. CHEM. 257, 13892 (1982) and PROC. NATL. ACAD. SCI. USA 75,4194 (1978)). Alternatively, liposomes containing dyes of the inventionhaving a lipophilic substituent (e.g. alkyl having 11-22 carbons),within their membranes are prepared by co-dissolving the fluorescentlipid and the unlabeled lipids phospholipid(s) that make up the liposomebefore forming the liposome dispersion essentially as described bySzoka, Jr. et al. (ANN. REV. BIOPHYS. BIOENG. 9, 467 (1980)).

Example 86 Preparation of Fluorescent Dye-Conjugates of Bacteria

Heat-killed Escherichia coli are suspended at 10 mg/mL in pH 8-9 bufferthen incubated with 0.5-1.0 mg/mL of an amine-reactive dye, typically asuccinimidyl ester derivative. After 30-60 minutes the labeled bacteriaare centrifuged and washed several times with buffer to remove anyunconjugated dye. Labeled bacteria that are opsonized are taken up bymacrophage, as determined by flow cytometry.

Example 87 Preparation of Nucleotide Dye-Conjugates

To 2 mg of 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (SigmaChemical) in 100 μL water is added Compound 9 or 69 in 100 μL DMF and 5μL triethylamine. After 3 hours, the solution is evaporated and theresidue is purified by HPLC. The product fractions are lyophilized togive the red-fluorescent nucleotide conjugate.

Alternatively, fluorescent dye-conjugates of deoxyuridine5′-triphosphate are prepared from 5-(3-amino-1-propynyl)-2′-deoxyuridine5′-triphosphate (as described in Hobbs, Jr. et al, supra), or bytreating a thiolated nucleotide or a thiophosphate nucleotide with athiol-reactive dye of the invention (such as the maleimide Compound 11).

Additionally, 2′-(or 3′)-2-aminoethylaminocarbonyladenosine5′-triphosphate is reacted with a slight excess of Compound 9 and,following precipitation with ethanol, the ribose-modified product ispurified by preparative HPLC.

Example 88 Preparation of an Oligonucleotide Dye-Conjugate

A 5′-amine-modified, 18-base M13 primer sequence (˜100 μg) is dissolvedin 4 μL H₂O. To this is added 250 μg of Compound 9 or 69 in 100 μL 0.1 Msodium borate, pH 8.5. After 16 hours, 10 μL of 5 M NaCl and 3 volumesof cold ethanol are added. The mixture is cooled to −20° C.,centrifuged, the supernatant is decanted, the pellet is rinsed withethanol and then dissolved in 100 μL H₂O. The labeled oligonucleotide ispurified by HPLC on a 300 Å C8 reverse-phase column using a rampgradient of 0.1 M triethylammonium acetate (pH˜7) and acetonitrile(5-95% over 30 min). The desired peak is collected and evaporated togive the fluorescent oligonucleotide.

Example 89 Preparing DNA Hybridization Probes Using FluorescentNucleotide Dye-Conjugates

For each labeling reaction, a microfuge tube containing about 1 μg of a˜700 bp Hind III-Bgl II fragment of the E. coli lacZ structural gene isheated for ˜10 minutes at 95° C. to fully separate the strands. The DNAis cooled on ice. A 2 μL aliquot of a 2 mg/mL mixture of random sequencehexanucleotides in 0.5 M Tris-HCl, pH 7.2, 0.1 M MgCl₂, 1 mMdithiothreitol is added, followed by 2 μL of a dNTP labeling mixture (1mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65 mM dTTP and 0.35 mM fluorescentdye-labeled dUTP (as prepared in Example 88). Sterile, distilled,deionized water is added to bring the total volume to 19 μL. 1 μL KlenowDNA polymerase (2 units/μL) is added. The samples are incubated 1 hr at37° C. The reactions are stopped with 2 μL of 0.2 M EDTA, pH 8.0. Thelabeled DNA is precipitated with 2.5 μL of 4 M LiCl and 75 μL of −20° C.ethanol. After 2 hours at −20° C. the precipitated nucleic acids arecentrifuged at 12,000 rpm. The pellets are washed with cold 70% ethanol,then cold 100% ethanol. The pellets are dried and dissolved in 10 mMTris-HCl, pH 8.0, 1 mM EDTA. A portion of each sample is analyzed by gelelectrophoresis on a 1% agarose minigel under standard conditions. Thelabeled DNA products are suitable for in situ hybridization experimentsfor the detection of RNA or DNA, such as is associated with the E. colilacZ gene in cells or tissues.

Example 90 Incorporation of Fluorescent Nucleotide Conjugates into DNAAmplification Products

A DNA amplification reaction is prepared as follows: 1 μL each of 20 μMsolutions of two oligonucleotide primers that hybridize to the humanβ-actin gene are added to a labeling reaction containing 5 μL DNAtemplate (100 μmol of a plasmid containing the entire gene), 5 μL 10×reaction buffer (100 mM Tris, pH 8.3, 500 mM KCl), 2.5 μL 1 mMfluorescent-labeled dUTP (Example 87), 1 μL 10 mM dATP, 1 μL 10 mM dCTP,1 μL 10 mM dGTP, 1.5 μL 5 mM dTTP, 3 μL 25 mM MgCl₂, and 28 μLdistilled, deionized water. The sample is transferred to a thermocyclerand processed as follows: one cycle, 94° C., 2.5 minutes; 30 cycles, 94°C., 1 minute, 50° C., 1 minute, 72° C., 1 minute; one cycle, 72° C., 5minutes; then 4° C. overnight. An aliquot of the sample is mixed with anequal volume of 10% glycerol, loaded onto a 0.9% agarose minigel andelectrophoresed. Fluorescent bands of the expected size are visible whenthe gel is illuminated with 300 nm ultraviolet light.

Example 91 In Situ Hybridization of an RNA Probe

Mouse fibroblasts are fixed and prepared for mRNA in situ hybridizationusing standard procedures. A dye-labeled RNA probe is prepared by invitro transcription of a plasmid containing the mouse actin structuralgene cloned downstream of a phage T3 RNA polymerase promoter. Labelingreactions comprise combining 2 μL DNA template (1 μg DNA), 1 μL each of10 mM ATP, CTP and GTP, 0.75 μL 10 mM UTP, 2.5 μL 1 mMaminoallyl-labeled UTP (Example 87), 2 μL 10× transcription buffer (400mM Tris, pH 8.0, 100 mM MgCl₂, 20 mM spermidine, 100 mM NaCl), 1 μL T3RNA polymerase (40 units/μL), 1 μL 2 mg/mL BSA, and 8.75 μL water.Reactions are incubated at 37° C. for two hours.

The DNA template is removed by treatment with 20 units DNase I for 15minutes, at 37° C. The RNA transcript is purified by extraction with anequal volume of phenol:chloroform, 1:1, then by chromatography onSEPHADEX G50. Labeled RNA is denatured for 5 minutes at 50° C., thenhybridized to cellular preparations using standard procedures. Thelong-wavelength fluorescence of the labeled cells is detected byexcitation through an optical filter optimized for Cy5-like dyes (OmegaXF47). The spatially integrated fluorescence from the FISH target region(18 separate intensities) as a function of the number of dyesincorporated per base of probe for conjugates of Compound 9 and of Cy5reveal that conjugates of Compound 9 are brighter than the correspondingCy5 probe by greater than 50% (at ˜13 bases per dye incorporation).

Example 92 Preparing DNA Hybridization Probes Using Fluorescent PlatinumDye (ULS)-Compounds

A fluorescent platinum complex (ULS) is prepared from a Compound of theinvention and from Cy5 monosuccinimidyl ester by adapting the methodsprovided in U.S. Pat. No. 5,714,327 to Houthoff et al. (1998) to yieldthe compound:

where FLUOR is any of the dye compounds of the invention, attached atits R³ substituent. For each labeling reaction, a microfuge tubecontaining 1 μg of pUC1.77 plasmid DNA containing a chromosome 1 humanα-satellite probe (DNase treated to a fragment size between 500-1000 bp)in 5 mM Tris, pH 8, 1 mM EDTA, is heated for ˜10 minutes at 95° C. tofully denature the DNA. The DNA is cooled on ice. 1 μL of a 1 mg/mLsolution of the prepared ULS complex is added, followed by the additionof 5 mM Tris, pH 8, 1 mM EDTA to bring the total volume to 25 μL. Thesamples are incubated 15 minutes at 80° C. The reactions are stopped onice. The labeled DNA is purified on a Bio-Rad Micro Bio-Spin P-30 TrisChromatography Column. The labeled DNA products are suitable for in situhybridization experiments.

A series of Compound 9 ULS and Cy5 ULS DNA hybridization probes areexamined with the number of dyes per base varying from 0 per 100 bases,to approximately 8 dyes per hundred bases (FIG. 11). Similar to thebehavior of the Cy5 bioconjugates of proteins (Examples 33, 34) and onaminoallyl-labeled DNA (Example 59), the fluorescently quenched 600 nmabsorbance band greatly increases at larger numbers of Cy5 dyes per DNAbase (equivalent to higher DOS in protein examples) (open circles, FIG.11), but does not increase with the Compound 9 ULS-labeled DNA (closedcircles, FIG. 11).

Example 93 Preparing DNA Hybridization Probes Using Amine-Modified DNAand an Amine-Reactive Dye of the Invention

Nick translation is performed using pUC1.77 plasmid DNA containing achromosome 1 human α-satellite probe. To a microcentrifuge tube isadded, in the following order: 23.5 μL H₂O, 5 μL 10× Nick Translationbuffer (0.5 M Tris-HCl, 50 mM MgCl₂, 0.5 mg/mL BSA, pH 7.8), 5 μL 0.1 MDTT, 4 μL d(GAC)TP mix (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP), 1 μL 0.5mM dTTP, 4 μL 0.5 mM aminoallyl-dUTP, 1 μL 1 μg/μL template DNA, 5 μLDNase I (1 μg/mL, 2000 Kunitz units/mg), 1.5 μL DNA polymerase I (10U/μL). The tube is incubated 2 hours at 15° C., then brought to a finalvolume of 100 μL with H₂O. The amine-modified DNA is purified using aQIAQUICK PCR purification Kit (Qiagen) with the following modificationsto purify the DNA from the enzyme and amine-containing Compounds: 75%EtOH is substituted for the wash buffer, H₂O is substituted for theelution buffer, and elution is performed twice for 5 minutes each. TheDNA is precipitated by adding 1/10 volume 3 M sodium acetate and 2.5volumes 100% EtOH, incubated at −70° C. for 30 minutes, centrifuged for15 minutes, and washed with 70% EtOH.

The amine-modified DNA is resuspended in 5 μL H₂O. To the solution isadded 3 μL 25 mg/mL sodium bicarbonate and 50 μg of Compound 9 or 69 in5 μL DMF. The reaction is incubated for 1 hour at room temperature inthe dark, to the reaction is added 90 μL H₂O, and it is purified using aQIAQUICK PCR purification kit (QIAGEN), with the followingmodifications: three washes are performed with 75% EtOH and threeelutions of 5 minutes each with the QIAGEN elution buffer. The DNA isprecipitated as before. The labeled DNA products are suitable for insitu hybridization experiments, use on microarrays and as fluorescencedonors or acceptors in hybridization-based assays. A comparison ofvarying the amino-allyl dUTP to dTTP ratios, followed by subsequentconjugation of the cDNA with either Compound 9 or Cy5 is shown in theTable 5. In the presence of excess reactive dye, both Compound 9 and Cy5derivatives can label cDNA to equivalent degrees.

TABLE 8 Nucleotide ratio, Compound 9 Cy5 AA-dUTP:dTTP bases/dyebases/dye 90 μM:10 μM 13.2 13.6 60 μM:10 μM 14.8 16.1 30 μM:10 μM 17.615 10 μM:10 μM 18.1 18.9  3 μM:10 μM 23.3 23.7  1 μM:10 μM 30.2 32.2

Example 94 Comparison of the Absorbance and Fluorescence Characteristicsof Nucleic Acids Prepared as in Example 93 from Compound 9 and from Cy5Monosuccinimidyl Ester

Nucleic acid conjugates of Compound 9 and of Cy5 monosuccinimidyl esterare prepared from the same batch of amine-substituted nucleic acid.Using the above protocol, it is possible to select a dye-labeling ratiothat is essentially identical for both the Cy5 derivative and theCompound 9 derivative (e.g., 1 dye per 23 to 24 bases, Table 5).Absorption spectra at the same nucleic acid concentration and dyelabeling ratio show a shifting of extinction from the long-wavelength650 nm band to a non-emitting 600 nm band for the Cy5 conjugate relativeto the conjugate of Compound 9 (FIG. 12). This result is very similar tothe absorbance changes observed for Cy5 conjugates of proteins (FIGS. 2,3). Fluorescence emission spectra excited at 600 nm reveal ˜4× greaterfluorescence of the conjugate of Compound 9 cDNA relative to the Cy5cDNA conjugate (FIG. 12).

To further prove that it is the behavior of the Cy5 when derivatizedonto the cDNA that causes the large increase in the 600 nm absorbanceband and decrease in the 650 nm absorbance, the synthesized Cy5 labeledcDNA is digested with micrococcal nuclease (EC 3.1.31.1; WorthingtonBiochemicals). The enzyme is dissolved at 1.0 mg/mL in reagent-gradewater and diluted to approximately 0.001 mg/mL in 0.1% bovine serumalbumin prior to addition to the Cy5 dye- and Compound 9-labeled cDNA's,pH=8.8, 0.1 M sodium borate, 0.01 M calcium chloride. The reaction isallowed to proceed at room temperature for 4 hours.

A comparison of the absorbance spectra of the identical labeled cDNAbefore and after treatment with the nuclease (which will digest the cDNAto dNMP's) is shown in FIG. 13. One can clearly see that the distortionin the absorbance of the Cy5-cDNA conjugate can be eliminated bydigesting the cDNA down to its individual deoxynucleosidemonophosphates.

There is a concomitant increase in the fluorescence of the Cy5 cDNA upondigestion of ˜15×, revealing that this absorbance pattern change isassociated with the large decrease in the fluorescence associated withthe Cy5-cDNA conjugate.

Example 95 Discrimination of Live and Dead Cells Using the Dyes of theInvention

Selected dyes of the invention are highly polar and therefore relativelyimpermeant to the membranes of live cells. These dyes can therefore beused to discriminate cells that have intact versus compromised cellmembranes in a single-color assay as follows: Mouse monocyte-macrophage,Abelson Leukemia Virus Transformed (RAW264.7) cells are trypsinized andwashed with PBS, pH 7.2. Approximately 8-10 million cells suspended in180 μL of PBS, pH 7.2 are placed in a glass test tube and heated in awater bath at 50° C. for 20 minutes to kill a fraction of the cells.Approximately 60 μL (2-3 million cells) of the cell suspension is addedto 940 μL of PBS, pH 7.2, followed by 0.1 μL of a 1 mg/mL solution of asuccinimidyl ester derivative of a dye of the invention in DMSO. Themixture is incubated on ice for 30 minutes and washed twice with PBS,followed by addition of 200 μL of PBS, pH 7.2. An identical aliquot ofcells is treated with 2 μL of a 150 μM solution of propidium iodide inwater (as a control for dead cells). Analysis of the cell suspensionusing flow cytometry shows that populations of dead cells stained by theinstant Compounds and those stained by propidium iodide are verysimilar.

Example 96 Staining Cells with Tandem Dye-Labeled Streptavidin

Jurkat cells are washed twice with 1% BSA/PBS and resuspended at aconcentration of 1×10⁷ cells/mL. The Jurkat cells are then incubated onice for 60 minutes with Mouse anti-Human CD4 Biotin (BiosourceInternational) at the recommended concentration of 10 μL for 1×10⁶cells. After incubation with the primary antibody, the cells are washedwith 1% BSA/PBS and incubated on ice for 30 minutes with 1 μg of eitherthe fluorescent streptavidin-phycoerythrin conjugate of Example 40, or astreptavidin conjugate of GIBCO'S RED 670. The cells are washed with 1%BSA/PBS, centrifuged, and resuspended with 400 μL of 1% BSA/PBS. Thesamples are analyzed on a FacsVantage flow cytometer exciting with the488-nm line of an argon laser, collecting the emission by a 700-nm longpass filter (XF-48). Using a FSC versus SSC dot plot the live cells aregated and the geometric mean of the fluorescence for FL3 is measured.The data is analyzed for both fluorescence and signal/noise ratio. Thecells stained with a conjugate of the invention exhibit asignal-to-noise ratio approximately 13% greater than those labeled withGIBCO RED 670.

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example, numerousmodifications, substitutions, and alterations are possible withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1. A method of staining a biological sample, comprising: combining a dyesolution comprising a compound having the formula:

and its salts, wherein R³ comprises an acrylamide, an activated ester ofa carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkylhalide, an amine, an anhydride, an aniline, an aryl halide, an azide, anaziridine, a boronate, a carboxylic acid, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazide, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, areactive platinum complex, a sulfonyl halide, a thiol group, a protein,a peptide, an amino acid, a dextran, a hormone, a lectin, alipopolysaccharide, a biological cell, a nucleotide, an oligonucleotide,or a small-molecule drug; R⁴ is a C₁-C₂₂ alkyl or C₇-C₂₂ arylalkyl, eachalkyl portion of which optionally incorporates up to six hetero atoms,selected from N, O and S, and each alkyl portion of which is optionallysubstituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo,phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C₁-C₆alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino, or C₃-C₁₈trialkylammonium; or R³ and R⁴ taken in combination complete a five- orsix-membered saturated or unsaturated ring that is substituted by agroup comprising an acrylamide, an activated ester of a carboxylic acid,an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine,an anhydride, an aniline, an aryl halide, an azide, an aziridine, aboronate, a carboxylic acid, a diazoalkane, a haloacetamide, ahalotriazine, a hydrazide, an imido ester, an isocyanate, anisothiocyanate, a maleimide, a phosphoramidite, a reactive platinumcomplex, a sulfonyl halide, a thiol group, a protein, a peptide, anamino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, abiological cell, a nucleotide, an oligonucleotide, or a small-moleculedrug; R² is C₁-C₂₂ alkyl or C₇-C₂₂ arylalkyl, each alkyl portion ofwhich optionally incorporates up to six hetero atoms, selected from N, Oand S, and each alkyl portion of which is optionally substituted one ormore times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino,sulfate, phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆alkylamino, C₂-C₁₂ dialkylamino, or C₃-C₁₈ trialkylammonium; R⁶ is H,amino, sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, or C₂-C₁₂ dialkylamino, each alkyl portion of which isoptionally further substituted by substituents selected from the groupconsisting of carboxy, sulfo, amino, and hydroxyl; R⁷ is H, amino,sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, or C₂-C₁₂ dialkylamino, each alkyl portion of which isoptionally further substituted by substituents selected from the groupconsisting of carboxy, sulfo, amino, and hydroxyl; R⁸ is H, amino,sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, or C₂-C₁₂ dialkylamino, each alkyl portion of which isoptionally further substituted by substituents selected from the groupconsisting of carboxy, sulfo, amino, and hydroxyl; R⁹ is H, amino,sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, or C₂-C₁₂ dialkylamino, each alkyl portion of which isoptionally further substituted by substituents selected from the groupconsisting of carboxy, sulfo, amino, and hydroxyl; or a memberindependently selected from R⁶ in combination with R⁷; R⁷ in combinationwith R⁸; and R⁸ in combination with R⁹; together with the atoms to whichthey are joined, form an aromatic ring comprising —CH or —CR¹ wherein R¹is amino, sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy,C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino, wherein each alkyl portion ofwhich is optionally substituted by substituents selected from the groupconsisting of carboxy, sulfo, amino, and hydroxyl; n is 0, 1, 2 or 3; Wrepresents the atoms necessary to form one to two fused aromatic ringshaving 5 or 6 atoms in each ring, wherein said W atoms are selected fromthe group consisting of —CH, —C, —CR^(1′), and —N(R¹²)_(β)′, where β′ is0 or 1, but no more than one of said atoms in W is —N(R¹²)_(β)′, andeach R^(1′) is amino, sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino, wherein eachalkyl portion of which is optionally substituted by substituentsselected from the group consisting of carboxy, sulfo, amino, andhydroxy; δ is 0 or 1, and δ+β′=1; R¹² is C₁-C₂₂ alkyl or C₇-C₂₂arylalkyl, each alkyl portion of which optionally incorporates up to sixhetero atoms, selected from the group consisting of N, O and S, and eachalkyl portion of which is optionally substituted one or more times by F,Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate,phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂dialkylamino, or C₃-C₁₈ trialkylammonium; R¹³ is C₁-C₂₂ alkyl or C₇-C₂₂arylalkyl, each alkyl portion of which optionally incorporates up to sixhetero atoms, selected from the group consisting of N, O and S, and eachalkyl portion of which is optionally substituted one or more times bysubstituents selected from the group consisting of F, Cl, Br, I,hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano,nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, andC₃-C₁₈ trialkylammonium; R¹⁴ is C₁-C₂₂ alkyl or C₇-C₂₂ arylalkyl, eachalkyl portion of which optionally incorporates up to six hetero atoms,selected from the group consisting of N, O and S, and each alkyl portionof which is optionally substituted one or more times by substituentsselected from the group consisting of F, Cl, Br, I, hydroxy, carboxy,sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido,C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, and C₃-C₁₈trialkylammonium, or R¹³ and R¹⁴ taken in combination complete a five-or six-membered saturated or unsaturated ring that is optionallysubstituted; with a biological sample in a concentration sufficient toyield a detectable optical response under desired conditions.
 2. Themethod according to claim 1, wherein R³ comprises a protein, a peptide,an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, abiological cell, a nucleotide, an oligonucleotide, or a small-moleculedrug.
 3. The method according to claim 1, wherein R³ comprises anacrylamide, an activated ester of a carboxylic acid, an acyl azide, anacyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, ananiline, an aryl halide, an azide, an aziridine, a boronate, acarboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, ahydrazide, an imido ester, an isocyanate, an isothiocyanate, amaleimide, a phosphoramidite, a reactive platinum complex, a sulfonylhalide, or a thiol group.
 4. The method according to claim 1, wherein R³comprises an antibody or fragment thereof, a blood component protein, ablood vessel proliferation inhibition factor, an enzyme, an enzymeinhibitor, a hormone, an IgG-binding protein, a fluorescent protein, agrowth factor, a metal-binding protein, a microorganism or portionthereof, a neuropeptide, a peptide toxin, a phospholipid-bindingprotein, or a structural protein.
 5. The method according to claim 1,wherein the sample comprises cells, proteins, or nucleic acid polymers.6. The method according to claim 1, wherein the sample is contained inor on a solid or semi-solid matrix that is a membrane, anelectrophoretic gel, silicon chip, a glass slide, a microwell plate or amicrofluidic chip.
 7. A method for forming a dye-conjugate of a protein,peptide or a nucleic acid polymer, said method comprising: combining adye solution comprising a compound of the formula

and its salts, wherein R³ comprises an acrylamide, an activated ester ofa carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkylhalide, an amine, an anhydride, an aniline, an aryl halide, an azide, anaziridine, a boronate, a carboxylic acid, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazide, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, areactive platinum complex, a sulfonyl halide, or a thiol group; R⁴ isC₁-C₂₂ alkyl or C₇-C₂₂ arylalkyl, each alkyl portion of which optionallyincorporates up to six hetero atoms, selected from N, O and S, and eachalkyl portion of which is optionally substituted one or more times by F,Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate,phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, orC₂-C₁₂ dialkylamino, or C₃-C₁₈ trialkylammonium; or R³ and R⁴ taken incombination complete a five- or six-membered saturated or unsaturatedring that is substituted by an acrylamide, an activated ester of acarboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkylhalide, an amine, an anhydride, an aniline, an aryl halide, an azide, anaziridine, a boronate, a carboxylic acid, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazide, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, areactive platinum complex, a sulfonyl halide, or a thiol group; R² isC₁-C₂₂ alkyl or C₇-C₂₂ arylalkyl, each alkyl portion of which optionallyincorporates up to six hetero atoms, selected from N, O and S, and eachalkyl portion of which is optionally substituted one or more times by F,Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate,phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂dialkylamino, or C₃-C₁₈ trialkylammonium; R⁶ is H, amino, sulfo,trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino,or C₂-C₁₂ dialkylamino, each alkyl portion of which is optionallyfurther substituted by substituents selected from the group consistingof carboxy, sulfo, amino, and hydroxyl; R⁷ is H, amino, sulfo,trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino,or C₂-C₁₂ dialkylamino, each alkyl portion of which is optionallyfurther substituted by substituents selected from the group consistingof carboxy, sulfo, amino, and hydroxyl; R⁸ is H, amino, sulfo,trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino,or C₂-C₁₂ dialkylamino, each alkyl portion of which is optionallyfurther substituted by substituents selected from the group consistingof carboxy, sulfo, amino, and hydroxyl; R⁹ is H, amino, sulfo,trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino,or C₂-C₁₂ dialkylamino, each alkyl portion of which is optionallyfurther substituted by substituents selected from the group consistingof carboxy, sulfo, amino, and hydroxyl; or a member independentlyselected from R⁶ in combination with R⁷; R⁷ in combination with R⁸; andR⁸ in combination with R⁹; together with the atoms to which they arejoined, form an aromatic ring comprising —CH or —CR¹ wherein R¹ isamino, sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, or C₂-C₁₂ dialkylamino, wherein each alkyl portion of whichis optionally substituted by substituents selected from the groupconsisting of carboxy, sulfo, amino, and hydroxyl; n is 0, 1, 2 or 3; Wrepresents the atoms necessary to form one to two fused aromatic ringshaving 5 or 6 atoms in each ring, wherein said W atoms are selected fromthe group consisting of —CH, —C, —CR^(1′), and —N(R¹²)_(β)′, where β′ is0 or 1, but no more than one of said atoms in W is —N(R¹²)_(β)′, andeach R^(1′) is amino, sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino, wherein eachalkyl portion of which is optionally substituted by substituentsselected from the group consisting of carboxy, sulfo, amino, andhydroxy; δ is 0 or 1, and δ+β′=1; R¹² is C₁-C₂₂ alkyl or C₇-C₂₂arylalkyl, each alkyl portion of which optionally incorporates up to sixhetero atoms, selected from the group consisting of N, O and S, and eachalkyl portion of which is optionally substituted one or more times by F,Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate,phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂dialkylamino, or C₃-C₁₈ trialkylammonium; R¹³ is C₁-C₂₂ alkyl armorC₇-C₂₂ arylalkyl, each alkyl portion of which optionally incorporates upto six hetero atoms, selected from the group consisting of N, O and S,and each alkyl portion of which is optionally substituted one or moretimes by substituents selected from the group consisting of F, Cl, Br,I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate,cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂dialkylamino, and C₃-C₁₈ trialkylammonium; R¹⁴ is C₁-C₂₂ alkyl or C₇-C₂₂arylalkyl, each alkyl portion of which optionally incorporates up to sixhetero atoms, selected from the group consisting of N, O and S, and eachalkyl portion of which is optionally substituted one or more times bysubstituents selected from the group consisting of F, Cl, Br, I,hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano,nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, andC₃-C₁₈ trialkylammonium, or R¹³ and R¹⁴ taken in combination complete afive- or six-membered saturated or unsaturated ring that is optionallysubstituted; with a sample comprising a protein, peptide or a nucleicacid polymer whereby a dye conjugate is formed.
 8. The method accordingto claim 7, wherein R³ comprises a carboxylic acid, an amine, anactivated ester of a carboxylic acid, a halotriazine, a hydrazine, amaleimide, or a reactive platinum complex.